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  • 91
    Millipore anti pak1
    A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity. ( a ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAP interaction between βPix and srGAP1. ( b ) Concurrent decreased association of βPix with known effector <t>Pak1</t> when migrating on fibrillar collagen. Blots are representative of three independent experiments. ( c ) RhoA activity determined by GST-RBD binding from NS and srGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagen environments. ( d ) Quantification of bands again revealed a 40-60% collagen-specific increase in RhoA activity after loss of srGAP1 (mean ± s.e.m., n = 3 independent western blots, t -tests). ( e ) srGAP1 knockdown HFFs were cultured overnight in 3D collagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded, protrusive (white arrowheads) morphology akin to βPix knockdown. Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiber arrangement (red, reflection microscopy) adjacent to the cell. Hole in matrix marked by white asterisk; scale bar, 25 μm. ( f ) Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3D collagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( g ) Quantification of cell velocity in cells treated with srGAP1 siRNA in 3D collagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). Statistical source data can be found in Supplementary Table 2 , *** P
    Anti Pak1, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc pak1
    <t>PAK1</t> positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
    Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology pak1
    PAK3 splice variants form heterodimers with <t>PAK1.</t> A , schematic representation of the PAK3 protein indicating the location of the b and c inserts. B , presence of inserts decreases dimer formation. All the PAK1 and PAK3 expressed proteins carry the kinase
    Pak1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pak1/product/Santa Cruz Biotechnology
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    99
    Cell Signaling Technology Inc anti pak1
    NDRG1 suppressed Rac1 activity and its downstream effector <t>PAK1</t> (A, B) A Rac1 activation assay was performed (see Materials and Methods ) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); * p
    Anti Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pak1  (Abcam)
    92
    Abcam pak1
    EOPK suppresses AKT and ERK phosphorylation and β-catenin expression via <t>PAK1</t> inhibition in HCT116 colorectal cancer cells. (A) HCT116 cells were treated with various concentrations of EOPK (25, 50, 100 μg/ml). (B) HCT116 cells were transfected with PAK1-siRNA or a control siRNA for 48 h and incubated in the presence or absence of EOPK (100 μg/ml) for 24 h. For both experiments, cell lysates were prepared and subjected to Western blotting to determine the expression of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, β-catenin and β-actin. Band density of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, and β-catenin was quantified using Gelpro analyzer (Media Cybernetics, Bethesda, MD, USA).
    Pak1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti pak1
    <t>Pak1</t> silencing partially restores adherens junctions. (A) Immunoblot analysis showing reduced Pak1 expression in Rb-/- MC3T3 cells infected with an adenovirus vector carrying an RNAi against Pak1, relative to a scrambled vector control. (B) Immunofluorescence labeling showing re-establishment of beta-catenin presence in the intercellular spaces after infection of Rb-/- MC3T3 cells with adeno-Pak1 RNAi ( top panel , arrow ), relative to Rb-/- MC3T3 cells infected with a scrambled control-carrying adenovirus ( middle panel ). Untreated Rb+/+ MC3T3 cells showing intercellular beta-catenin labeling are shown for comparison ( bottom panel , arrow ).
    Anti Pak1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc rabbit anti pak1
    Disruption of <t>Pak1</t> sensitizes the myocardium to I/R-induced ventricular arrhythmias.
    Rabbit Anti Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc pak1 2 3
    Dominant-negative PID expression inhibits cell viability and PAK phosphorylation in human MM cell lines. A) Immunoblot analysis of <t>P-PAK1/2/3,</t> total PAK1/2/3 and β-actin levels 72-hr post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22
    Pak1 2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti pak1
    Nischarin interacts with <t>PAK1/2</t> and regulates PAK activity. Lysates (500 μl, concentration 3 μg/μl) of rat cortical neurons were incubated with an anti-rabbit PAK1 polyclonal antibody, an anti-rabbit PAK2 polyclonal antibody or an anti-mouse Nischarin monoclonal antibody and their IgG then precipitated with G-Sepharose protein beads. (A) The Nischarin immunoprecipitates were blotted with anti-PAK1 antibody (top panel) or with anti-PAK2 antibody (bottom panel). (B) PAK1 or PAK2 immunoprecipitates were blotted with anti-Nischarin antibody. IgG was used as the negative control and 5% cell lysates (input) was added in the gel in each experiment to be a positive control. IP EX: immunoprecipitation extract. (C) Immunofluorescence staining was performed on primary cultured hippocampal neurons (10 DIV). Strong staining for Nischarin (green) co-localized both with PAK1 (red, left panel) and PAK2 (red, right panel) in the perinuclear region and the dendrites (arrows). Scale bars: 10 μm. (D, E) Nischarin-shRNA effectively suppresses the expression of Nischarin protein and stimulates the phosphorylation of both PAK1 and PAK2, in Neuro-2a cells (n = 3). Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (* p
    Anti Pak1, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Horizon Discovery pak1
    Group-I PAKs induce direct phosphorylation of HACE1. ( a ) In vitro [γ 32 P]-ATP kinase assay using recombinant 6His-HACE1 and recombinant <t>6His-PAK1</t> analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. ( b ) MCF12A and HUVEC protein lysates analyzed by immunoblot (IB) using anti-PAK1 and anti-PAK2 antibodies. IB: actin is used as a loading control. ( c–e ) Protein lysates from ( c ) HUVECs and ( d,e ) MCF12A cells transfected with siRNAs targeting ( c , d ) PAK2 or ( e ) PAK1 and plasmid expressing HA-HACE1 and myc-Rac1(Q61L) and analyzed by IB using the indicated antibodies. ( f ) Protein lysates from MCF12A cells transfected with HA-HACE1, either intoxicated with CNF1 for 16 hours or co-transfected with myc-Rac1(Q61L), and treated with FRAX597 at the indicated concentration for 16 hours before IB analysis. ( g , h ) Protein lysates from MCF12A cells transfected with HA-HACE1, myc-Rac1(Q61L) and GST-KID2 or Flag-PAK1K141A and analyzed by IB. IBs in ( a – h .
    Pak1, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genentech pak1 inhibitor
    <t>PAK1</t> activation is correlated with tumor stage at presentation. A -Dot plot demonstrating the expression of E-cadherin in the examined tumors in relation to the surgical stage of each tumor. The correlation between variables were examined by Spearman Rank Correlation analysis. B - Dot plot demonstrating the expression of p-PAK1(Thr423) in stage I and stage II/III tumors. The mean between groups was compared by student T-test. C - Dot plot demonstrating the expression of p-Crk-II(Ser41) in stage I and stage II/III tumors. The mean between groups was compared by student T-test.
    Pak1 Inhibitor, supplied by Genentech, used in various techniques. Bioz Stars score: 91/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc pak1 pbd agarose
    <t>PAK1</t> activation is correlated with tumor stage at presentation. A -Dot plot demonstrating the expression of E-cadherin in the examined tumors in relation to the surgical stage of each tumor. The correlation between variables were examined by Spearman Rank Correlation analysis. B - Dot plot demonstrating the expression of p-PAK1(Thr423) in stage I and stage II/III tumors. The mean between groups was compared by student T-test. C - Dot plot demonstrating the expression of p-Crk-II(Ser41) in stage I and stage II/III tumors. The mean between groups was compared by student T-test.
    Pak1 Pbd Agarose, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 89/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pak1
    Both PIX and NCK localize to focal adhesions in breast cancer cell lines with active <t>PAK1/2.</t> SK-BR-3 and ZR-75-1 cells were fixed and stained with anti-PIX/anti-rabbit Alexa Fluor 568 or anti-NCK/anti-rabbit Alexa Fluor 488 as described in MATERIALS AND METHODS. Bar, 10 μm.
    Pak1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti pak1 2 3
    Both PIX and NCK localize to focal adhesions in breast cancer cell lines with active <t>PAK1/2.</t> SK-BR-3 and ZR-75-1 cells were fixed and stained with anti-PIX/anti-rabbit Alexa Fluor 568 or anti-NCK/anti-rabbit Alexa Fluor 488 as described in MATERIALS AND METHODS. Bar, 10 μm.
    Anti Pak1 2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity. ( a ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAP interaction between βPix and srGAP1. ( b ) Concurrent decreased association of βPix with known effector Pak1 when migrating on fibrillar collagen. Blots are representative of three independent experiments. ( c ) RhoA activity determined by GST-RBD binding from NS and srGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagen environments. ( d ) Quantification of bands again revealed a 40-60% collagen-specific increase in RhoA activity after loss of srGAP1 (mean ± s.e.m., n = 3 independent western blots, t -tests). ( e ) srGAP1 knockdown HFFs were cultured overnight in 3D collagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded, protrusive (white arrowheads) morphology akin to βPix knockdown. Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiber arrangement (red, reflection microscopy) adjacent to the cell. Hole in matrix marked by white asterisk; scale bar, 25 μm. ( f ) Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3D collagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( g ) Quantification of cell velocity in cells treated with srGAP1 siRNA in 3D collagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). Statistical source data can be found in Supplementary Table 2 , *** P

    Journal: Nature cell biology

    Article Title: An extracellular matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration

    doi: 10.1038/ncb3026

    Figure Lengend Snippet: A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity. ( a ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAP interaction between βPix and srGAP1. ( b ) Concurrent decreased association of βPix with known effector Pak1 when migrating on fibrillar collagen. Blots are representative of three independent experiments. ( c ) RhoA activity determined by GST-RBD binding from NS and srGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagen environments. ( d ) Quantification of bands again revealed a 40-60% collagen-specific increase in RhoA activity after loss of srGAP1 (mean ± s.e.m., n = 3 independent western blots, t -tests). ( e ) srGAP1 knockdown HFFs were cultured overnight in 3D collagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded, protrusive (white arrowheads) morphology akin to βPix knockdown. Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiber arrangement (red, reflection microscopy) adjacent to the cell. Hole in matrix marked by white asterisk; scale bar, 25 μm. ( f ) Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3D collagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( g ) Quantification of cell velocity in cells treated with srGAP1 siRNA in 3D collagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). Statistical source data can be found in Supplementary Table 2 , *** P

    Article Snippet: Antibodies The anti-βPix antibody (07-1450, 1:1000), anti-GFP (3F8.2, 1:1000), anti-PAK1 (EP656Y, 1:500), anti-Rac3 (07-2151, 1:500), anti-Rac2 (07-604, 1:500), and anti-PPPR2A1 (07-250, 1:1000) were from Millipore.

    Techniques: Activity Assay, Immunoprecipitation, Binding Assay, Western Blot, Cell Culture, Labeling, Microscopy

    PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Inhibition, shRNA

    PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Migration, Over Expression, Transfection, Plasmid Preparation, Transwell Invasion Assay, Microscopy

    PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: In Vitro, In Vivo, Kinase Assay, Glutathione S-Transferase Assay, Staining, Purification, Transfection, Incubation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Plasmid Preparation, Fluorescence, Expressing

    The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. ( a ) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. ( b ) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. ( c ) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed > 50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues ( P =0.003). ( d ) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from ( b ). Spearman's correlation coefficient is 0.661 ( P =0.002). ( e ) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μ m

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: The positive correlation between RUFY3 and PAK1 in gastric cancer cells and clinical gastric cancer tissue samples. ( a ) The protein levels of RUFY3 in gastric cancer cell lines (BGC-823, MKN45, AGS MGC-803, SGC-7901, BGC-823 and MKN1) relative to the normal gastric epithelial cell line (GES-1) were analyzed by western blot. ( b ) The protein level of RUFY3 is positively correlated with PAK1 in gastric cancers and matched adjacent normal gastric tissue samples. Total protein from gastric cancer samples was extracted, and the protein levels of RUFY3 and PAK1 were measured by western blot. ( c ) Histogram showed the relative level of RUFY3 in gastric cancer tissues. Among 40 patients with gastric cancer, 28 of 40 (70%) samples revealed > 50% increase in the RUFY3 level relative to their matched non-tumor adjacent tissues ( P =0.003). ( d ) The relative protein level of RUFY3 was plotted against that of PAK1 in gastric cancers tissue samples with Spearman's correlation statistical analysis from ( b ). Spearman's correlation coefficient is 0.661 ( P =0.002). ( e ) Immunohistochemical analyses of RUFY3 and PAK1 expression in adjacent normal gastric tissues and metastatic gastric cancer. Scale bar, 50 μ m

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Western Blot, Immunohistochemistry, Expressing

    PAK1 expression was correlated with CK19, while PAK4 expression was correlated with α-SMA and palladin. The expression of multiple proteins in six TKCC cell lines was determined by Western blot (A), and results were analysed and quantified (B) as described in Materials and Methods. The intracellular localization of CK19, α-SMA were determined by immunofluorescent staining in two selected TKCC cell lines (TKCC 15, 18) (C). The fluorescent images were taken using a Nikon fluorescent microscope at × 40 magnification. The correlations between PAK1 or PAK4 expression with the expression of CK19, α-SMA and palladin were assessed by regression analysis, and R 2 and p values were presented (D). pPAK1, phospho-PAK1; tPAK1, total PAK1; pPAK4, phospho-PAK4; tPAK4, total PAK4; CK19, cytokeratin 19; α-SMA, alpha-smooth muscle actin.

    Journal: American Journal of Translational Research

    Article Title: PAK inhibition by PF-3758309 enhanced the sensitivity of multiple chemotherapeutic reagents in patient-derived pancreatic cancer cell lines

    doi:

    Figure Lengend Snippet: PAK1 expression was correlated with CK19, while PAK4 expression was correlated with α-SMA and palladin. The expression of multiple proteins in six TKCC cell lines was determined by Western blot (A), and results were analysed and quantified (B) as described in Materials and Methods. The intracellular localization of CK19, α-SMA were determined by immunofluorescent staining in two selected TKCC cell lines (TKCC 15, 18) (C). The fluorescent images were taken using a Nikon fluorescent microscope at × 40 magnification. The correlations between PAK1 or PAK4 expression with the expression of CK19, α-SMA and palladin were assessed by regression analysis, and R 2 and p values were presented (D). pPAK1, phospho-PAK1; tPAK1, total PAK1; pPAK4, phospho-PAK4; tPAK4, total PAK4; CK19, cytokeratin 19; α-SMA, alpha-smooth muscle actin.

    Article Snippet: The expression of total and phospho-PAK1 and PAK4 varied in the panel of six TKCC cell lines.

    Techniques: Expressing, Western Blot, Staining, Microscopy

    EGFR signaling in response to glutamine depletion drives macropinocytosis via Pak1. (A, B) Immunoblots assessing phosphorylation of Pak1/2 [p-Pak1 (S199/204)/p-Pak2 (S192/197)] in AsPC-1 cells under the indicated conditions. EGF treatment (A) was done at 100 ng/mL for 5 min. Erlotinib treatment (B) was done at the indicated concentrations for 2 hours. β-actin or α-tubulin was used as a loading control. The p-Pak/Pak1 ratios are shown. (C) Representative images of macropinocytosis (green) in the indicated cells treated with vehicle (DMSO) or FRAX597 (FRAX) in glutamine-free media. Scale bar, 20 μm. (D) Quantification of macropinocytosis in the indicated cells treated with DMSO in glutamine-containing media and under the conditions described in (C). Data are presented relative to the values obtained for the +Q/+DMSO condition and representative of at least 3 independent experiments. Error bars represent s.e.m. of n=3 replicates with at least 650 cells scored per condition. (E) Immunohistochemical staining of p-EGFR and total EGFR protein expression in AsPC-1 xenograft tumors treated with vehicle control or erlotinib. The inset boxes in the low-magnification images indicate the areas shown in the high-magnification images. The arrows indicate the tumor peripheral regions, and the arrowheads indicate the tumor non-peripheral regions. Black scale bar, 200 μm; red scale bar, 20 μm. (F) Quantification of macropinocytosis in AsPC-1 xenograft tumors treated with vehicle control (Ctrl) or erlotinib (ERL). Error bars represent s.e.m. of n=3 tumors. (G) Representative images from sections of AsPC-1 xenograft tumors treated with vehicle control or erlotinib with macropinosomes labeled with TMR-dextran (red) and tumor cells immunostained with anti-CK8 (green). Nuclei are labeled with DAPI (blue). Scale bar, 20 μm. (H) Immunofluorescence staining in AsPC-1 xenograft tumors treated with vehicle control or erlotinib. Tumor sections were stained with anti-CK8 (red), anti-p-Pak1 (green) and DAPI (blue). Scale bar, 20 μm. .

    Journal: Developmental cell

    Article Title: EGFR/Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis

    doi: 10.1016/j.devcel.2019.05.043

    Figure Lengend Snippet: EGFR signaling in response to glutamine depletion drives macropinocytosis via Pak1. (A, B) Immunoblots assessing phosphorylation of Pak1/2 [p-Pak1 (S199/204)/p-Pak2 (S192/197)] in AsPC-1 cells under the indicated conditions. EGF treatment (A) was done at 100 ng/mL for 5 min. Erlotinib treatment (B) was done at the indicated concentrations for 2 hours. β-actin or α-tubulin was used as a loading control. The p-Pak/Pak1 ratios are shown. (C) Representative images of macropinocytosis (green) in the indicated cells treated with vehicle (DMSO) or FRAX597 (FRAX) in glutamine-free media. Scale bar, 20 μm. (D) Quantification of macropinocytosis in the indicated cells treated with DMSO in glutamine-containing media and under the conditions described in (C). Data are presented relative to the values obtained for the +Q/+DMSO condition and representative of at least 3 independent experiments. Error bars represent s.e.m. of n=3 replicates with at least 650 cells scored per condition. (E) Immunohistochemical staining of p-EGFR and total EGFR protein expression in AsPC-1 xenograft tumors treated with vehicle control or erlotinib. The inset boxes in the low-magnification images indicate the areas shown in the high-magnification images. The arrows indicate the tumor peripheral regions, and the arrowheads indicate the tumor non-peripheral regions. Black scale bar, 200 μm; red scale bar, 20 μm. (F) Quantification of macropinocytosis in AsPC-1 xenograft tumors treated with vehicle control (Ctrl) or erlotinib (ERL). Error bars represent s.e.m. of n=3 tumors. (G) Representative images from sections of AsPC-1 xenograft tumors treated with vehicle control or erlotinib with macropinosomes labeled with TMR-dextran (red) and tumor cells immunostained with anti-CK8 (green). Nuclei are labeled with DAPI (blue). Scale bar, 20 μm. (H) Immunofluorescence staining in AsPC-1 xenograft tumors treated with vehicle control or erlotinib. Tumor sections were stained with anti-CK8 (red), anti-p-Pak1 (green) and DAPI (blue). Scale bar, 20 μm. .

    Article Snippet: The following primary antibodies were used: p-PAK1 (Ser144)/p-PAK2 (Ser141) (1:100) (CST, 2606), PAK1 (1:50) (Santa Cruz, sc-882), and CK8 (1:1000) (DSHB, TROMA-I).

    Techniques: Western Blot, Immunohistochemistry, Staining, Expressing, Labeling, Immunofluorescence

    PAK3 splice variants form heterodimers with PAK1. A , schematic representation of the PAK3 protein indicating the location of the b and c inserts. B , presence of inserts decreases dimer formation. All the PAK1 and PAK3 expressed proteins carry the kinase

    Journal: The Journal of Biological Chemistry

    Article Title: The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity *

    doi: 10.1074/jbc.M112.355073

    Figure Lengend Snippet: PAK3 splice variants form heterodimers with PAK1. A , schematic representation of the PAK3 protein indicating the location of the b and c inserts. B , presence of inserts decreases dimer formation. All the PAK1 and PAK3 expressed proteins carry the kinase

    Article Snippet: Altogether, our results indicate that, in transfected cells, the two proteins PAK1 and PAK3 localized and that PAK3a preferentially forms heterodimers with PAK1.

    Techniques:

    PAK3 and PAK1 are coexpressed in single neurons, colocalize in dendritic spines, co-purified with the postsynaptic density, and co-immunoprecipitate in brain extracts. A , schematic representation of the location of the different PAK3 oligonucleotides

    Journal: The Journal of Biological Chemistry

    Article Title: The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity *

    doi: 10.1074/jbc.M112.355073

    Figure Lengend Snippet: PAK3 and PAK1 are coexpressed in single neurons, colocalize in dendritic spines, co-purified with the postsynaptic density, and co-immunoprecipitate in brain extracts. A , schematic representation of the location of the different PAK3 oligonucleotides

    Article Snippet: Altogether, our results indicate that, in transfected cells, the two proteins PAK1 and PAK3 localized and that PAK3a preferentially forms heterodimers with PAK1.

    Techniques: Purification

    PAK3a forms heterodimers with PAK1. A , PAK1 and PAK3 colocalize at focal adhesions of HeLa cells. FLAG-PAK3a-KD and HA-PAK1-KD plasmids were transfected in HeLa cells. Immunolabeling was performed using FLAG and HA antibodies to detect PAK proteins and

    Journal: The Journal of Biological Chemistry

    Article Title: The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity *

    doi: 10.1074/jbc.M112.355073

    Figure Lengend Snippet: PAK3a forms heterodimers with PAK1. A , PAK1 and PAK3 colocalize at focal adhesions of HeLa cells. FLAG-PAK3a-KD and HA-PAK1-KD plasmids were transfected in HeLa cells. Immunolabeling was performed using FLAG and HA antibodies to detect PAK proteins and

    Article Snippet: Altogether, our results indicate that, in transfected cells, the two proteins PAK1 and PAK3 localized and that PAK3a preferentially forms heterodimers with PAK1.

    Techniques: Transfection, Immunolabeling

    PAK1 regulates PAK3 activity through heterodimerization. A , control of PAK expression. HA-tagged PAK3a-WT and PAK3b-WT plasmids were transfected in HeLa cells, and cell lysates were HA-immunoprecipitated ( IP HA ), then the amount of PAK3 proteins was determined

    Journal: The Journal of Biological Chemistry

    Article Title: The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity *

    doi: 10.1074/jbc.M112.355073

    Figure Lengend Snippet: PAK1 regulates PAK3 activity through heterodimerization. A , control of PAK expression. HA-tagged PAK3a-WT and PAK3b-WT plasmids were transfected in HeLa cells, and cell lysates were HA-immunoprecipitated ( IP HA ), then the amount of PAK3 proteins was determined

    Article Snippet: Altogether, our results indicate that, in transfected cells, the two proteins PAK1 and PAK3 localized and that PAK3a preferentially forms heterodimers with PAK1.

    Techniques: Activity Assay, Expressing, Transfection, Immunoprecipitation

    NDRG1 suppressed Rac1 activity and its downstream effector PAK1 (A, B) A Rac1 activation assay was performed (see Materials and Methods ) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); * p

    Journal: Oncotarget

    Article Title: The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1

    doi:

    Figure Lengend Snippet: NDRG1 suppressed Rac1 activity and its downstream effector PAK1 (A, B) A Rac1 activation assay was performed (see Materials and Methods ) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); * p

    Article Snippet: Anti-human primary antibodies were implemented at a 1:1, 000–2, 000 dilution and include: anti-NDRG1 (Cat#:ab37897) from Abcam (Cambridge); anti-Src (Cat#:2123), anti-phospho-Src Family (Tyr416; Cat.#:6943), anti-phospho-Src(Tyr527; Cat.#:2105), anti-p130Cas (Cat.#:13846), anti-phospho-p130Cas (Tyr249; Cat.#:4014), anti-phospho-p130Cas (Tyr410; Cat.#:4011), anti-EGF Receptor (Cat.#:2926), anti-phospho-EGF Receptor (Tyr1148; Cat.#:4404), anti-c-Abl (Cat.#:2862), anti-phospho-c-Abl (Tyr245; Cat.#:2861), anti-PAK1 (Cat.#:2602), anti-phospho-PAK1 (Thr423; Cat.#:2606), anti-PTP-PEST (Cat.#:4864), anti-PTP1B (Cat.#:5311), anti-CrkII (Cat.#:3492) and anti-phospho-CrkII (Tyr221; Cat.#:3491) were from Cell Signaling Technology (Boston, MA); and anti-Rac1 (Cat.#:05–389) was from Millipore (Darmstadt).

    Techniques: Activity Assay, Activation Assay, Expressing

    Effects of wild-type GIT1 and GIT1-R283W on neuronal PAK activation and GAD1 expression. ( A-E ) Primary cultured hippocampal neurons at DIV 14–16 were infected with lentiviruses expressing the indicated wild-type GIT1, GIT1-R283W, or empty vector control (Puro) constructs. One week later, neurons were harvested in 2X SDS sample buffer and subjected to immunoblot analysis using the indicated antibodies ( A ). ( B ) Immunoblot intensities for PAK3, PAK1, and phospho-PAK (S144, S199/S204, or T423) were normalized to GAPDH values, and then compared to empty vector control lentivirus values to obtain fold changes. Significant elevation of pPAK (S199/S204) and pPAK (T423) levels occurred with expression of wild-type GIT1 but not GIT1-R283W. Data represent mean + SEM; N = 4; **** = p

    Journal: Molecular psychiatry

    Article Title: Functional Analysis of Rare Variants Found in Schizophrenia Implicates a Critical Role for GIT1-PAK3 Signaling in Neuroplasticity

    doi: 10.1038/mp.2016.98

    Figure Lengend Snippet: Effects of wild-type GIT1 and GIT1-R283W on neuronal PAK activation and GAD1 expression. ( A-E ) Primary cultured hippocampal neurons at DIV 14–16 were infected with lentiviruses expressing the indicated wild-type GIT1, GIT1-R283W, or empty vector control (Puro) constructs. One week later, neurons were harvested in 2X SDS sample buffer and subjected to immunoblot analysis using the indicated antibodies ( A ). ( B ) Immunoblot intensities for PAK3, PAK1, and phospho-PAK (S144, S199/S204, or T423) were normalized to GAPDH values, and then compared to empty vector control lentivirus values to obtain fold changes. Significant elevation of pPAK (S199/S204) and pPAK (T423) levels occurred with expression of wild-type GIT1 but not GIT1-R283W. Data represent mean + SEM; N = 4; **** = p

    Article Snippet: anti-phospho-AKT (Ser473) (Cell Signaling, #4058), anti-phosphop44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, #9101), anti-phospho-GRIA1 (Ser845) (Millipore, #04–1073), anti-phospho-MEK1 (MAPK2K1) (Ser298) (Cell Signaling, #9128), anti-phospho-PAK1 (Ser144)/PAK2 (Ser141)/PAK3 (Ser139) (Cell Signaling, #2606), anti-phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197)/PAK3 (Ser200/205) (Cell Signaling, #2605), anti-phospho-PAK1 (Thr423)/PAK2 (Thr402)/PAK3 (Thr421) (Cell Signaling, #2601), anti-phospho-SRC Family (Tyr416) (Cell Signaling, #2101).).

    Techniques: Activation Assay, Expressing, Cell Culture, Infection, Plasmid Preparation, Construct

    Pak1 regulation of p41-Arc phosphorylation. ( A ) MCF-7 cells were co-transfected with T7–p41-Arc along with Pak1 autoinhibitory domain (GST–Pak1-83-149) Pak1, wild type (wt) activated Pak1 T423E or activated Rac1 (V12Rac1). After 48 h, cells were metabolically labelled with 32 P-orthophosphate and treated with or without 100 ng/ml EGF for 5 min. In vivo phosphorylation of T7–p41-Arc was assessed by immunoprecipitation. Western blot with GST antibody was used to analyse the expression of GST–Pak1-83-149, and c-myc tag antibody was used to analyse the expression of Pak1 and Rac1 constructs. ( B ) Pak1 phosphorylates p41-Arc. Purified Arp2/3 complex from platelet cells was used as a substrate in in vitro Pak1 kinase assays. Reaction components were separated by SDS–PAGE, transferred to a nylon membrane and autoradiographed. Blots were probed using p41-Arc and p34 antibodies. ( C ) Serum-starved MCF-7 cells were metabolically labelled with 32 P-orthophosphate, treated with EGF (100 ng/ml, 2 min), and endogenous p41-Arc was immunoprecipitated using polyclonal antibody raised against it (kindly provided by Dr Mathew Welch). ( D ) Pak1 phosphorylates p41-Arc in vitro . Pak1 kinase assay was performed using purified Pak1 enzyme and p41-Arc as a substrate. MBP was used as a positive control. ( E ) Mapping of Pak1 phosphorylation site in p41-Arc. In vitro phosphorylation of GST fusion proteins of p41-Arc deletion mutants by Pak1 enzyme (middle panel). Ponceau stain of the GST p41-Arc fusion proteins used in the assay (bottom panel). Autophosphorylation of Pak1 is shown in the top panel. ( F ) Phosphorylation of GST-p41-Arc T21A or GST-p41-Arc T69A mutants by Pak1 enzyme. ( G ) MCF-7 cells were co-transfected with p41-Arc wt or p41-Arc T21A. Cells were metabolically labelled with 32 P-orthophosphate, treated with or without EGF, and in vivo phosphorylation of p41-Arc constructs was analysed.

    Journal: EMBO Reports

    Article Title: p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate

    doi: 10.1038/sj.embor.7400079

    Figure Lengend Snippet: Pak1 regulation of p41-Arc phosphorylation. ( A ) MCF-7 cells were co-transfected with T7–p41-Arc along with Pak1 autoinhibitory domain (GST–Pak1-83-149) Pak1, wild type (wt) activated Pak1 T423E or activated Rac1 (V12Rac1). After 48 h, cells were metabolically labelled with 32 P-orthophosphate and treated with or without 100 ng/ml EGF for 5 min. In vivo phosphorylation of T7–p41-Arc was assessed by immunoprecipitation. Western blot with GST antibody was used to analyse the expression of GST–Pak1-83-149, and c-myc tag antibody was used to analyse the expression of Pak1 and Rac1 constructs. ( B ) Pak1 phosphorylates p41-Arc. Purified Arp2/3 complex from platelet cells was used as a substrate in in vitro Pak1 kinase assays. Reaction components were separated by SDS–PAGE, transferred to a nylon membrane and autoradiographed. Blots were probed using p41-Arc and p34 antibodies. ( C ) Serum-starved MCF-7 cells were metabolically labelled with 32 P-orthophosphate, treated with EGF (100 ng/ml, 2 min), and endogenous p41-Arc was immunoprecipitated using polyclonal antibody raised against it (kindly provided by Dr Mathew Welch). ( D ) Pak1 phosphorylates p41-Arc in vitro . Pak1 kinase assay was performed using purified Pak1 enzyme and p41-Arc as a substrate. MBP was used as a positive control. ( E ) Mapping of Pak1 phosphorylation site in p41-Arc. In vitro phosphorylation of GST fusion proteins of p41-Arc deletion mutants by Pak1 enzyme (middle panel). Ponceau stain of the GST p41-Arc fusion proteins used in the assay (bottom panel). Autophosphorylation of Pak1 is shown in the top panel. ( F ) Phosphorylation of GST-p41-Arc T21A or GST-p41-Arc T69A mutants by Pak1 enzyme. ( G ) MCF-7 cells were co-transfected with p41-Arc wt or p41-Arc T21A. Cells were metabolically labelled with 32 P-orthophosphate, treated with or without EGF, and in vivo phosphorylation of p41-Arc constructs was analysed.

    Article Snippet: Antibodies against Pak1 were purchased from Cell signaling and Zymed Inc. Antibodies for myc tag, the T7 tag and phalloidin were from MBL Laboratories, Novagen and Molecular Probes, respectively.

    Techniques: Transfection, Metabolic Labelling, In Vivo, Immunoprecipitation, Western Blot, Expressing, Construct, Purification, In Vitro, SDS Page, Kinase Assay, Positive Control, Staining

    Pak1 interaction with p41-Arc. ( A ) p41-Arc was translated in vitro and the 35 S-labelled protein was incubated with either GST or GST-Pak1 and interaction was analysed by GST pull-down assays. Input represents 1/20th of the total 35 S-labelled p41-Arc used in the assay. ( B ) 35 S-labelled Pak1 was incubated with GST or GST-p41-Arc, and binding was analysed by GST pull-down assays. guanosine 5′-[γ-thio] triphosphate (GTPγS)-loaded GST-v12Rac was used as a positive control. Input represents 1/20th of the total 35 S-labelled Pak1 used in the assay. ( C ) Crosslinking analysis of Pak1 to p41-Arc. Purified GST-Pak1 was incubated with an increasing amount of in vitro -translated 35 S-labelled p41-Arc and crosslinked using a zero-length crosslinker EDC/NHC. Crosslinked products were denatured by heating in SDS buffer and separated on an SDS–PAGE gel followed by autoradiography. ( D ) In vivo interaction of Pak1 and p41-Arc. Total cell lysate from MCF-7 cells growing in 10% serum (2 mg) was immunoprecipitated with either IgG- or a p41-Arc-specific antibody, blotted and probed with antibodies specific for Pak1, p41-Arc and p34. ( E ) MCF-7 cells were transfected with both myc-tagged Pak1 and immunostained with antibodies against myc tag (green) and endogenous p41-Arc (red), and with phalloidin staining for F-actin (blue). Arrows indicate points of white colour indicating three-colour colocalization.

    Journal: EMBO Reports

    Article Title: p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate

    doi: 10.1038/sj.embor.7400079

    Figure Lengend Snippet: Pak1 interaction with p41-Arc. ( A ) p41-Arc was translated in vitro and the 35 S-labelled protein was incubated with either GST or GST-Pak1 and interaction was analysed by GST pull-down assays. Input represents 1/20th of the total 35 S-labelled p41-Arc used in the assay. ( B ) 35 S-labelled Pak1 was incubated with GST or GST-p41-Arc, and binding was analysed by GST pull-down assays. guanosine 5′-[γ-thio] triphosphate (GTPγS)-loaded GST-v12Rac was used as a positive control. Input represents 1/20th of the total 35 S-labelled Pak1 used in the assay. ( C ) Crosslinking analysis of Pak1 to p41-Arc. Purified GST-Pak1 was incubated with an increasing amount of in vitro -translated 35 S-labelled p41-Arc and crosslinked using a zero-length crosslinker EDC/NHC. Crosslinked products were denatured by heating in SDS buffer and separated on an SDS–PAGE gel followed by autoradiography. ( D ) In vivo interaction of Pak1 and p41-Arc. Total cell lysate from MCF-7 cells growing in 10% serum (2 mg) was immunoprecipitated with either IgG- or a p41-Arc-specific antibody, blotted and probed with antibodies specific for Pak1, p41-Arc and p34. ( E ) MCF-7 cells were transfected with both myc-tagged Pak1 and immunostained with antibodies against myc tag (green) and endogenous p41-Arc (red), and with phalloidin staining for F-actin (blue). Arrows indicate points of white colour indicating three-colour colocalization.

    Article Snippet: Antibodies against Pak1 were purchased from Cell signaling and Zymed Inc. Antibodies for myc tag, the T7 tag and phalloidin were from MBL Laboratories, Novagen and Molecular Probes, respectively.

    Techniques: In Vitro, Incubation, Binding Assay, Positive Control, Purification, SDS Page, Autoradiography, In Vivo, Immunoprecipitation, Transfection, Staining

    Pak1 regulation of the p41-Arc interaction with the Arp2/3 complex in vivo . ( A ) MCF-7 cells were transfected with control or Pak1-specific siRNA, metabolically labelled with 32 P-orthophosphate, and treated with EGF (2 min), and phosphorylation of p41-Arc was measured by immunoprecipitation. An aliquot of total lysate was analysed by western blotting for expression of Pak1. Vinculin was used as a loading control. ( B , C ) MCF-7 cells were transfected with T7-tagged wt p41-Arc, T7-p41-Arc T21A or T7-p41-Arc T21E, serum starved for 48 h, and then treated with 1 nM EGF for 30 min before processing for immunostaining of T7 (red), endogenous p34 (green) and F-actin (blue). Additional experimental groups were co-transfected with either Pak1-specific siRNA or with control noninhibitory siRNA, serum starved for 48 h, and then stimulated with EGF. ( B ) Examples of p34 and p41-Arc colocalization in control and Pak1 siRNA-treated cells. ( C ) The mean (±s.d.) percentage of cells with focal cytoplasmic or membranous colocalization points was assessed for each group ( n =250 cells per group). Data are mean±s.d. of three experiments. ( D ) MCF-7 cells were transfected with or without activated Rac1 (V12Rac1) to activate Pak1. Pak1 was immunoprecipitated from cell lysates and analysed for Arp2/3 complex proteins. ( E ) MCF-7 cells were transfected with T7-p41-Arc, serum starved for 48 h, treated with EGF for 2 min, and cell lysates were immunoprecipitated with anti-T7 antibody and analysed for the presence of Arp complex proteins by western blotting. ( F ) MCF-7 cells were transfected with wt, T21E or T21A mutants of T7-p41-Arc. After 72 h, cell lysates were immunoprecipitated with anti-T7 antibody and analysed for the presence of Arp complex proteins by western blotting. ( G ) MCF-7 cells were transfected with either T21A or T21E mutants of T7-p41-Arc. F-actin levels in these cells were analysed by confocal microscopy. The percentage of transfected cells ( n =100) showing greater overall pixel intensity of phalloidin taining than neighbouring untransfected cells as quantified using Zeiss image analysis software.

    Journal: EMBO Reports

    Article Title: p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate

    doi: 10.1038/sj.embor.7400079

    Figure Lengend Snippet: Pak1 regulation of the p41-Arc interaction with the Arp2/3 complex in vivo . ( A ) MCF-7 cells were transfected with control or Pak1-specific siRNA, metabolically labelled with 32 P-orthophosphate, and treated with EGF (2 min), and phosphorylation of p41-Arc was measured by immunoprecipitation. An aliquot of total lysate was analysed by western blotting for expression of Pak1. Vinculin was used as a loading control. ( B , C ) MCF-7 cells were transfected with T7-tagged wt p41-Arc, T7-p41-Arc T21A or T7-p41-Arc T21E, serum starved for 48 h, and then treated with 1 nM EGF for 30 min before processing for immunostaining of T7 (red), endogenous p34 (green) and F-actin (blue). Additional experimental groups were co-transfected with either Pak1-specific siRNA or with control noninhibitory siRNA, serum starved for 48 h, and then stimulated with EGF. ( B ) Examples of p34 and p41-Arc colocalization in control and Pak1 siRNA-treated cells. ( C ) The mean (±s.d.) percentage of cells with focal cytoplasmic or membranous colocalization points was assessed for each group ( n =250 cells per group). Data are mean±s.d. of three experiments. ( D ) MCF-7 cells were transfected with or without activated Rac1 (V12Rac1) to activate Pak1. Pak1 was immunoprecipitated from cell lysates and analysed for Arp2/3 complex proteins. ( E ) MCF-7 cells were transfected with T7-p41-Arc, serum starved for 48 h, treated with EGF for 2 min, and cell lysates were immunoprecipitated with anti-T7 antibody and analysed for the presence of Arp complex proteins by western blotting. ( F ) MCF-7 cells were transfected with wt, T21E or T21A mutants of T7-p41-Arc. After 72 h, cell lysates were immunoprecipitated with anti-T7 antibody and analysed for the presence of Arp complex proteins by western blotting. ( G ) MCF-7 cells were transfected with either T21A or T21E mutants of T7-p41-Arc. F-actin levels in these cells were analysed by confocal microscopy. The percentage of transfected cells ( n =100) showing greater overall pixel intensity of phalloidin taining than neighbouring untransfected cells as quantified using Zeiss image analysis software.

    Article Snippet: Antibodies against Pak1 were purchased from Cell signaling and Zymed Inc. Antibodies for myc tag, the T7 tag and phalloidin were from MBL Laboratories, Novagen and Molecular Probes, respectively.

    Techniques: In Vivo, Transfection, Metabolic Labelling, Immunoprecipitation, Western Blot, Expressing, Immunostaining, Confocal Microscopy, Software

    Rac1–PAK1/2–cofilin signaling and the F-actin/G-actin ratio are increased in FXS model mice ( A to H ) Representative western blots and quantification assessing the abundance of (A) total Rac1 relative to tubulin (G), (B) Rac1-GTP abundance by PAK-PBD pulldown ( t = 3.44), (C) phospho-Ser 199/204 PAK1 and Ser 192/197 PAK2 relative to total PAK1/2 (E; t = 3.31), (D) phospho-Ser 3 cofilin relative to total cofilin ( t = 2.24), (F) cofilin, and (H) the F-actin/G-actin ratio ( t = 2.51) in the hippocampal CA1 area of FXS model mice and wild-type littermates. Data are mean ± SEM normalized to wild-type controls. N = 4-10 samples per genotype; not significant unless noted; * p

    Journal: Science signaling

    Article Title: Reducing eIF4E-eIF4G Interactions Restores the Balance Between Protein Synthesis and Actin Dynamics in Fragile X Syndrome Model Mice

    doi: 10.1126/scisignal.aan0665

    Figure Lengend Snippet: Rac1–PAK1/2–cofilin signaling and the F-actin/G-actin ratio are increased in FXS model mice ( A to H ) Representative western blots and quantification assessing the abundance of (A) total Rac1 relative to tubulin (G), (B) Rac1-GTP abundance by PAK-PBD pulldown ( t = 3.44), (C) phospho-Ser 199/204 PAK1 and Ser 192/197 PAK2 relative to total PAK1/2 (E; t = 3.31), (D) phospho-Ser 3 cofilin relative to total cofilin ( t = 2.24), (F) cofilin, and (H) the F-actin/G-actin ratio ( t = 2.51) in the hippocampal CA1 area of FXS model mice and wild-type littermates. Data are mean ± SEM normalized to wild-type controls. N = 4-10 samples per genotype; not significant unless noted; * p

    Article Snippet: The following antibodies were used in the western blot experiments: phospho-PAK1(Ser199/204)/PAK2(Ser192/197), PAK1/2, phospho-cofilin (Ser3), cofilin (1:500; Cell Signaling Technology) and tubulin (1:5000; Cell Signaling Technology), which was used to determine the total amounts of the proteins.

    Techniques: Mouse Assay, Western Blot

    Rac1–PAK1/2–cofilin signaling pathway is dysregulated in FXS model mice but is normalized by blocking the interaction of eIF4E and eIF4G ( A to E ) Representative western blots and quantification of (A) phospho-Ser 199/204 /Ser 192/197 PAK1/2 and (B) phospho-Ser 3 cofilin, relative to (D) total PAK1/2 or (E) total cofilin, respectively, and of tubulin (C, loading control), in area CA1 of hippocampal slices obtained from wild-type (WT) or FXS mice treated with DHPG (50 μM for 10 min), 4EGI-1 (100 μM; alone or applied 40 min before DHPG), or the combination DHPG and 4EGI-1. N = 9-22 (WT) or 11-17 (FXS) samples per treatment. Data are mean ± SEM normalized to wild-type vehicle-treated controls. Effects of DHPG treatment [F (1, 47) =4.76 (A), F (1, 48) =7.12 (B)], 4EGI-1 treatment [F (1, 47) =5.3 (A), F (1, 48) =5.06 (B)], and DHPG+4EGI-1 treatment [F (1, 47) =5.4 (A), F (1, 48) =6.5 (B)] in samples from WT mice were significant (analyzed by two-way ANOVA, each p

    Journal: Science signaling

    Article Title: Reducing eIF4E-eIF4G Interactions Restores the Balance Between Protein Synthesis and Actin Dynamics in Fragile X Syndrome Model Mice

    doi: 10.1126/scisignal.aan0665

    Figure Lengend Snippet: Rac1–PAK1/2–cofilin signaling pathway is dysregulated in FXS model mice but is normalized by blocking the interaction of eIF4E and eIF4G ( A to E ) Representative western blots and quantification of (A) phospho-Ser 199/204 /Ser 192/197 PAK1/2 and (B) phospho-Ser 3 cofilin, relative to (D) total PAK1/2 or (E) total cofilin, respectively, and of tubulin (C, loading control), in area CA1 of hippocampal slices obtained from wild-type (WT) or FXS mice treated with DHPG (50 μM for 10 min), 4EGI-1 (100 μM; alone or applied 40 min before DHPG), or the combination DHPG and 4EGI-1. N = 9-22 (WT) or 11-17 (FXS) samples per treatment. Data are mean ± SEM normalized to wild-type vehicle-treated controls. Effects of DHPG treatment [F (1, 47) =4.76 (A), F (1, 48) =7.12 (B)], 4EGI-1 treatment [F (1, 47) =5.3 (A), F (1, 48) =5.06 (B)], and DHPG+4EGI-1 treatment [F (1, 47) =5.4 (A), F (1, 48) =6.5 (B)] in samples from WT mice were significant (analyzed by two-way ANOVA, each p

    Article Snippet: The following antibodies were used in the western blot experiments: phospho-PAK1(Ser199/204)/PAK2(Ser192/197), PAK1/2, phospho-cofilin (Ser3), cofilin (1:500; Cell Signaling Technology) and tubulin (1:5000; Cell Signaling Technology), which was used to determine the total amounts of the proteins.

    Techniques: Mouse Assay, Blocking Assay, Western Blot

    Optimisation of the PaRac1 for the synaptic application a, Isothermal titration calorimetry (ITC) experiments showing that the introduction of L514K and L531E mutations into the original PaRac1 construct 12 reduced binding with the CRIB domain of PAK1 in the dark. The light-insensitive form of LOV2 (C450A) and the I539E mutant, which mimics the unfolded ‘lit state’, were used as negative and positive controls, respectively. b , Leaky activity of PaRac1 in the dark. In hippocampal neuronal cultures transfected with the original PaRac1, we observed a bearded appearance of the soma with a numerous ectopic dendrites, while neurons transfected with PaRac1 (L514K/L531E) were indistinguishable from normal neurons. c , Assessment of the affinity of PaRac1 to the endogenous PAK1 using a pull-down assay. HEK293 cells, which were transfected with PaRac1-Venus, were divided into two groups: lit and dark. The cells in the lit group were radiated with light with a white fluorescent lamp before cell lysis, and continuous light illumination was present during subsequent immunoprecipitation until the final wash step of protein precipitants. Conversely, cells in the dark group were lit with a yellow fluorescence lamp, which excludes light wavelengths below 500 nm. Co-immunoprecipitation with PAK1 revealed that PaRac1 (L514K/L531E) barely bound with PAK1 in the dark (The number of trial is depicted in the bar graph, ** P

    Journal: Nature

    Article Title: Labelling and optical erasure of synaptic memory traces in the motor cortex

    doi: 10.1038/nature15257

    Figure Lengend Snippet: Optimisation of the PaRac1 for the synaptic application a, Isothermal titration calorimetry (ITC) experiments showing that the introduction of L514K and L531E mutations into the original PaRac1 construct 12 reduced binding with the CRIB domain of PAK1 in the dark. The light-insensitive form of LOV2 (C450A) and the I539E mutant, which mimics the unfolded ‘lit state’, were used as negative and positive controls, respectively. b , Leaky activity of PaRac1 in the dark. In hippocampal neuronal cultures transfected with the original PaRac1, we observed a bearded appearance of the soma with a numerous ectopic dendrites, while neurons transfected with PaRac1 (L514K/L531E) were indistinguishable from normal neurons. c , Assessment of the affinity of PaRac1 to the endogenous PAK1 using a pull-down assay. HEK293 cells, which were transfected with PaRac1-Venus, were divided into two groups: lit and dark. The cells in the lit group were radiated with light with a white fluorescent lamp before cell lysis, and continuous light illumination was present during subsequent immunoprecipitation until the final wash step of protein precipitants. Conversely, cells in the dark group were lit with a yellow fluorescence lamp, which excludes light wavelengths below 500 nm. Co-immunoprecipitation with PAK1 revealed that PaRac1 (L514K/L531E) barely bound with PAK1 in the dark (The number of trial is depicted in the bar graph, ** P

    Article Snippet: The precipitate was immunoblotted with an anti-PAK1 antibody (#2602; Cell Signaling, Beverly, MA).

    Techniques: Isothermal Titration Calorimetry, Construct, Binding Assay, Mutagenesis, Activity Assay, Transfection, Pull Down Assay, Lysis, Immunoprecipitation, Fluorescence

    EOPK suppresses AKT and ERK phosphorylation and β-catenin expression via PAK1 inhibition in HCT116 colorectal cancer cells. (A) HCT116 cells were treated with various concentrations of EOPK (25, 50, 100 μg/ml). (B) HCT116 cells were transfected with PAK1-siRNA or a control siRNA for 48 h and incubated in the presence or absence of EOPK (100 μg/ml) for 24 h. For both experiments, cell lysates were prepared and subjected to Western blotting to determine the expression of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, β-catenin and β-actin. Band density of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, and β-catenin was quantified using Gelpro analyzer (Media Cybernetics, Bethesda, MD, USA).

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells

    doi: 10.1186/1472-6882-14-275

    Figure Lengend Snippet: EOPK suppresses AKT and ERK phosphorylation and β-catenin expression via PAK1 inhibition in HCT116 colorectal cancer cells. (A) HCT116 cells were treated with various concentrations of EOPK (25, 50, 100 μg/ml). (B) HCT116 cells were transfected with PAK1-siRNA or a control siRNA for 48 h and incubated in the presence or absence of EOPK (100 μg/ml) for 24 h. For both experiments, cell lysates were prepared and subjected to Western blotting to determine the expression of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, β-catenin and β-actin. Band density of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, and β-catenin was quantified using Gelpro analyzer (Media Cybernetics, Bethesda, MD, USA).

    Article Snippet: PAK1 was expressed in colorectal cancer cell lines including Colon26L5, HCT116, HCT15, HT29, and SW620 cells.

    Techniques: Expressing, Inhibition, Transfection, Incubation, Western Blot

    PAK1 siRNA enhances the inhibitory effect of EOPK on the cell proliferation and migration. (A) HCT116 cells were transfected with PAK1-siRNA for 48 h and were incubated in the presence or absence of EOPK (100 μg/ml) for 24 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of Cyclin D1, CDK4, CDK6 and β-actin. (B) HCT116 cells were transfected with PAK1-siRNA for 24 h and treated with EOPK (100 μg/ml) for 5 days. The cells were resolved in 70% ethanol after washing with distilled water, and crystal violet absorbance was read using a microplate reader. Data represent mean ± SD of three independent experiments. * p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells

    doi: 10.1186/1472-6882-14-275

    Figure Lengend Snippet: PAK1 siRNA enhances the inhibitory effect of EOPK on the cell proliferation and migration. (A) HCT116 cells were transfected with PAK1-siRNA for 48 h and were incubated in the presence or absence of EOPK (100 μg/ml) for 24 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of Cyclin D1, CDK4, CDK6 and β-actin. (B) HCT116 cells were transfected with PAK1-siRNA for 24 h and treated with EOPK (100 μg/ml) for 5 days. The cells were resolved in 70% ethanol after washing with distilled water, and crystal violet absorbance was read using a microplate reader. Data represent mean ± SD of three independent experiments. * p

    Article Snippet: PAK1 was expressed in colorectal cancer cell lines including Colon26L5, HCT116, HCT15, HT29, and SW620 cells.

    Techniques: Migration, Transfection, Incubation, Western Blot, Expressing

    PAK1 mediates MEK/ERK, PI3K/AKT, and Wnt/β-catenin in colon cancer cells. (A) Basal levels of PAK1 expression in colorectal cancer cell lines (human and murine) and normal colon cells extracted from mouse tissue were determined by Western blotting. (B) HCT116 cells were transfected with PAK1-siRNA1, PAK1-siRNA2, or control siRNA for 48 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, β-catenin and β-actin. (C) Various human colorectal cancer cells were treated with EOPK (100 μg/ml) for 24 h. (D) Cytotoxicity of EOPK was analyzed by MTT assay in NIH-3 T3 cells as a normal cell control.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells

    doi: 10.1186/1472-6882-14-275

    Figure Lengend Snippet: PAK1 mediates MEK/ERK, PI3K/AKT, and Wnt/β-catenin in colon cancer cells. (A) Basal levels of PAK1 expression in colorectal cancer cell lines (human and murine) and normal colon cells extracted from mouse tissue were determined by Western blotting. (B) HCT116 cells were transfected with PAK1-siRNA1, PAK1-siRNA2, or control siRNA for 48 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of PAK1, p-PAK1, AKT, p-AKT, ERK, p-ERK, β-catenin and β-actin. (C) Various human colorectal cancer cells were treated with EOPK (100 μg/ml) for 24 h. (D) Cytotoxicity of EOPK was analyzed by MTT assay in NIH-3 T3 cells as a normal cell control.

    Article Snippet: PAK1 was expressed in colorectal cancer cell lines including Colon26L5, HCT116, HCT15, HT29, and SW620 cells.

    Techniques: Expressing, Western Blot, Transfection, MTT Assay

    Pak1 silencing partially restores adherens junctions. (A) Immunoblot analysis showing reduced Pak1 expression in Rb-/- MC3T3 cells infected with an adenovirus vector carrying an RNAi against Pak1, relative to a scrambled vector control. (B) Immunofluorescence labeling showing re-establishment of beta-catenin presence in the intercellular spaces after infection of Rb-/- MC3T3 cells with adeno-Pak1 RNAi ( top panel , arrow ), relative to Rb-/- MC3T3 cells infected with a scrambled control-carrying adenovirus ( middle panel ). Untreated Rb+/+ MC3T3 cells showing intercellular beta-catenin labeling are shown for comparison ( bottom panel , arrow ).

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Pak1 silencing partially restores adherens junctions. (A) Immunoblot analysis showing reduced Pak1 expression in Rb-/- MC3T3 cells infected with an adenovirus vector carrying an RNAi against Pak1, relative to a scrambled vector control. (B) Immunofluorescence labeling showing re-establishment of beta-catenin presence in the intercellular spaces after infection of Rb-/- MC3T3 cells with adeno-Pak1 RNAi ( top panel , arrow ), relative to Rb-/- MC3T3 cells infected with a scrambled control-carrying adenovirus ( middle panel ). Untreated Rb+/+ MC3T3 cells showing intercellular beta-catenin labeling are shown for comparison ( bottom panel , arrow ).

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Expressing, Infection, Plasmid Preparation, Immunofluorescence, Labeling

    Conserved regions in the Pak1 promoter contain E2F binding sites. (A) Alignment of a region of 1,000 bp (from -700 to +300 bp) of the mouse and human Pak1 promoters generated by the Data Base of Transcriptional Start Sites (DBTSS), showing 7 conserved regions labeled 0–6. The longest conserved stretch is region 0, which spans a 406-bp region from -186 to +220 with 66% identity. The table below the promoter diagram shows the start and stop positions for each conserved region in mouse (m) and human (h). (B) Schematic of the human and mouse Pak1 promoters showing 5 E2F1 binding sites (labeled as 1–3 in the human promoter and 4 and 5 in the mouse promoter), as identified by Genomatrix analysis. These E2F1 binding sites are in the conserved region (labeled as 0 in A) . The table shows the positions, strand, and sequences of each E2F1 binding site, with the core nucleotides in each binding site indicated capitalized in bold. (C) A Pak1 mouse promoter-Firefly luciferase construct containing the 2 E2F binding sites was transfected into Rb+/+ and Rb-/- MC3T3 cells, and promoter activation was measured by its luciferase activity and normalized against a co-transfected Renilla luciferase construct. The Pak1 promoter region containing the E2F binding sites was amplified using the forward (F) and reverse (R) primers illustrated in (B) and in bold in S1 Fig . Transcriptional activity in Rb-/- cells was stronger than in Rb+/+ cells by a factor of 2.1 after normalization with Renilla luciferase, a value that is close to the 2.7-fold transcriptional induction that we observed in our nuclear run-on assays. *P

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Conserved regions in the Pak1 promoter contain E2F binding sites. (A) Alignment of a region of 1,000 bp (from -700 to +300 bp) of the mouse and human Pak1 promoters generated by the Data Base of Transcriptional Start Sites (DBTSS), showing 7 conserved regions labeled 0–6. The longest conserved stretch is region 0, which spans a 406-bp region from -186 to +220 with 66% identity. The table below the promoter diagram shows the start and stop positions for each conserved region in mouse (m) and human (h). (B) Schematic of the human and mouse Pak1 promoters showing 5 E2F1 binding sites (labeled as 1–3 in the human promoter and 4 and 5 in the mouse promoter), as identified by Genomatrix analysis. These E2F1 binding sites are in the conserved region (labeled as 0 in A) . The table shows the positions, strand, and sequences of each E2F1 binding site, with the core nucleotides in each binding site indicated capitalized in bold. (C) A Pak1 mouse promoter-Firefly luciferase construct containing the 2 E2F binding sites was transfected into Rb+/+ and Rb-/- MC3T3 cells, and promoter activation was measured by its luciferase activity and normalized against a co-transfected Renilla luciferase construct. The Pak1 promoter region containing the E2F binding sites was amplified using the forward (F) and reverse (R) primers illustrated in (B) and in bold in S1 Fig . Transcriptional activity in Rb-/- cells was stronger than in Rb+/+ cells by a factor of 2.1 after normalization with Renilla luciferase, a value that is close to the 2.7-fold transcriptional induction that we observed in our nuclear run-on assays. *P

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Binding Assay, Generated, Labeling, Luciferase, Construct, Transfection, Activation Assay, Activity Assay, Amplification

    Changes in Pak1 expression occur in multiple solid tumors. (A–J) GSE19188 data sets for E2F1 (A) and Pak1 (B) expression in NSCLC tumor and adjacent normal samples were ranked from most normal to most tumor-like. Samples 1–58 are the adjacent normal tissues (black diamonds), whereas samples 59–145 represent tumor samples (light gray diamonds). Gene expression was determined via microarray for E2F1 (C, G, E, I) and Pak1 (D, F, H, J) . Oncomine ( http://www.oncomine.org , Compendia Bioscience, Ann Arbor, MI, USA) was used for analysis and visualization of E2F1 and Pak1 expression in 4 common solid tumor types. (C, D) Analysis of E2F1 (C) and Pak1 (D) expression in normal lung versus squamous cell lung carcinoma from the Bhattacharjee lung database. (E, F) Analysis of E2F1 (E) and Pak1 (F) expression in normal lung versus squamous cell lung carcinoma from the Hou lung database. (G, H) Analysis of E2F1 (G) and Pak1 (H) expression in normal lung versus lung adenocarcinoma from the Stearman lung database. (I, J) Analysis of E2F1 (I) and Pak1 (J) expression in normal breast versus invasive breast carcinoma from the TCGA breast database.

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Changes in Pak1 expression occur in multiple solid tumors. (A–J) GSE19188 data sets for E2F1 (A) and Pak1 (B) expression in NSCLC tumor and adjacent normal samples were ranked from most normal to most tumor-like. Samples 1–58 are the adjacent normal tissues (black diamonds), whereas samples 59–145 represent tumor samples (light gray diamonds). Gene expression was determined via microarray for E2F1 (C, G, E, I) and Pak1 (D, F, H, J) . Oncomine ( http://www.oncomine.org , Compendia Bioscience, Ann Arbor, MI, USA) was used for analysis and visualization of E2F1 and Pak1 expression in 4 common solid tumor types. (C, D) Analysis of E2F1 (C) and Pak1 (D) expression in normal lung versus squamous cell lung carcinoma from the Bhattacharjee lung database. (E, F) Analysis of E2F1 (E) and Pak1 (F) expression in normal lung versus squamous cell lung carcinoma from the Hou lung database. (G, H) Analysis of E2F1 (G) and Pak1 (H) expression in normal lung versus lung adenocarcinoma from the Stearman lung database. (I, J) Analysis of E2F1 (I) and Pak1 (J) expression in normal breast versus invasive breast carcinoma from the TCGA breast database.

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Expressing, Microarray

    Rb does not affect Pak1 mRNA or protein stability. (A) Rb+/+ and Rb-/- MC3T3 cells were incubated with the transcriptional inhibitor actinomycin D, and Pak1 mRNA levels were assessed by qRT-PCR at 0, 5, 8, 10, 12, 17, 20, and 24 hours after actinomycin D addition. The graph shows percent of Pak1 mRNA, normalized against β-actin mRNA, remaining as a function of time incubated in actinomycin D relative to T 0 (no actinomycin D), which was arbitrarily assigned a value of 100%. Data were analyzed by nonlinear regression, and the Pak1 mRNA t 1/2 was calculated from the first-order decay constant ( k ) obtained with the PRISM software program (GraphPad). Rb+/+ and Rb-/- MC3T3 cells show similar decay constants ( k ) and t 1/2 and comparable to the previously reported 10 hours [ 21 ], suggesting that these parameters are not affected by Rb status. (B) Samples of total RNA from Rb+/+ and Rb-/- cells were collected at various time intervals (T1 –T6) during the time course of the experiment and run in a 1.4% agarose gel containing formaldehyde. Ethidium bromide staining revealed a 2:1 ratio of 28S-to-18S rRNA and no visible signs of degradation, indicating high quality of the RNA without any signs of degradation and suggesting that the measured degradation of Pak1 mRNA is not a reflection of a general degradation of total RNA. (C) Kinetics of decay of the myeloid cell leukemia-1 (MCL1) mRNA used as a reference. The experiment was performed essentially as described in (A) . In both Rb+/+ and Rb-/- MC3T3 cells, MCL1 mRNA had a t 1/2 of approximately 3 hours, which is what has been reported for this mRNA [ 22 ], and the k was unaffected by Rb status. (D) Immunoblot analysis of Pak1 protein levels at different time points after addition of 10 mg/mL puromycin ( top ) and graph showing changes of Pak1 protein expression levels as a function of time incubated in puromycin ( bottom ). The time points analyzed were 0, 2, 5, 8, and 11 hours. Drug toxicity precluded the analysis of time points beyond 11 hours. Pak1 levels were normalized against α-tubulin expression. No significant changes were observed in terms of Pak1 protein degradation when comparing Rb+/+ and Rb-/- MC3T3 cells, suggesting that Rb does not affect Pak1 protein stability, at least within the time points evaluated.

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Rb does not affect Pak1 mRNA or protein stability. (A) Rb+/+ and Rb-/- MC3T3 cells were incubated with the transcriptional inhibitor actinomycin D, and Pak1 mRNA levels were assessed by qRT-PCR at 0, 5, 8, 10, 12, 17, 20, and 24 hours after actinomycin D addition. The graph shows percent of Pak1 mRNA, normalized against β-actin mRNA, remaining as a function of time incubated in actinomycin D relative to T 0 (no actinomycin D), which was arbitrarily assigned a value of 100%. Data were analyzed by nonlinear regression, and the Pak1 mRNA t 1/2 was calculated from the first-order decay constant ( k ) obtained with the PRISM software program (GraphPad). Rb+/+ and Rb-/- MC3T3 cells show similar decay constants ( k ) and t 1/2 and comparable to the previously reported 10 hours [ 21 ], suggesting that these parameters are not affected by Rb status. (B) Samples of total RNA from Rb+/+ and Rb-/- cells were collected at various time intervals (T1 –T6) during the time course of the experiment and run in a 1.4% agarose gel containing formaldehyde. Ethidium bromide staining revealed a 2:1 ratio of 28S-to-18S rRNA and no visible signs of degradation, indicating high quality of the RNA without any signs of degradation and suggesting that the measured degradation of Pak1 mRNA is not a reflection of a general degradation of total RNA. (C) Kinetics of decay of the myeloid cell leukemia-1 (MCL1) mRNA used as a reference. The experiment was performed essentially as described in (A) . In both Rb+/+ and Rb-/- MC3T3 cells, MCL1 mRNA had a t 1/2 of approximately 3 hours, which is what has been reported for this mRNA [ 22 ], and the k was unaffected by Rb status. (D) Immunoblot analysis of Pak1 protein levels at different time points after addition of 10 mg/mL puromycin ( top ) and graph showing changes of Pak1 protein expression levels as a function of time incubated in puromycin ( bottom ). The time points analyzed were 0, 2, 5, 8, and 11 hours. Drug toxicity precluded the analysis of time points beyond 11 hours. Pak1 levels were normalized against α-tubulin expression. No significant changes were observed in terms of Pak1 protein degradation when comparing Rb+/+ and Rb-/- MC3T3 cells, suggesting that Rb does not affect Pak1 protein stability, at least within the time points evaluated.

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Incubation, Quantitative RT-PCR, Software, Agarose Gel Electrophoresis, Staining, Expressing

    Model linking Rb function to cell adhesion via control of Pak1 expression. (A) Rb-expressing osteoblasts show significantly diminished Pak1 expression relative to their Rb-deficient counterparts since Rb binds to E2F1 in the Pak1 promoter and blocks its activity. This can be either by direct interference with E2F1’s trans-activating capacity or by recruitment of histone deacetylases to E2F1-sensitive promoters ( 1 ). (B) In the absence of Rb, the unrestricted E2F1 action induces transcription of the Pak1 gene ( 1 ) with consequent translation of the Pak1 protein ( 2 ). Once translated, Pak1 binds and is activated by a Rho GTPase such as Rac1 or Cdc42 to form an active complex (3), which then phosphorylates the merlin tumor suppressor in serine 518 ( 4 ), a phosphorylation that impairs merlin function. Because merlin acts mainly by promoting the stabilization of adherens junctions at the cell membrane, loss of merlin function by the Pak1-dependent phosphorylation will bring about a disruption of adherens junctions and therefore of intercellular adhesion.

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Model linking Rb function to cell adhesion via control of Pak1 expression. (A) Rb-expressing osteoblasts show significantly diminished Pak1 expression relative to their Rb-deficient counterparts since Rb binds to E2F1 in the Pak1 promoter and blocks its activity. This can be either by direct interference with E2F1’s trans-activating capacity or by recruitment of histone deacetylases to E2F1-sensitive promoters ( 1 ). (B) In the absence of Rb, the unrestricted E2F1 action induces transcription of the Pak1 gene ( 1 ) with consequent translation of the Pak1 protein ( 2 ). Once translated, Pak1 binds and is activated by a Rho GTPase such as Rac1 or Cdc42 to form an active complex (3), which then phosphorylates the merlin tumor suppressor in serine 518 ( 4 ), a phosphorylation that impairs merlin function. Because merlin acts mainly by promoting the stabilization of adherens junctions at the cell membrane, loss of merlin function by the Pak1-dependent phosphorylation will bring about a disruption of adherens junctions and therefore of intercellular adhesion.

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Expressing, Activity Assay

    Rb represses the transcription rate of the Pak1 gene. (A) Representative nuclear run-on assay from a triplicate experiment showing hybridization of Rb+/+ ( blot 1 ) and Rb-/- ( blot 2 ) DIG-labeled mRNA probes to a Pak1 cDNA immobilized in a membrane. These two probes were also hybridized to GAPDH cDNAs ( blots 3 and 4 ). Hybridization signal intensity was stronger with the Rb-/- probe, indicating higher transcriptional rate of the Pak1 gene in Rb-/- nuclei. (B) To determine the differences in signal intensity, the chemioluminiscence in the membranes was quantified and analyzed using BioRad’s Quantity 1 Imaging Software and normalized against the signal intensities obtained when hybridizing to the GAPDH cDNAs (blots 3 and 4 in A ). This quantitative analysis showed a statistically significant 2.7-fold increase in the signal intensity of the Rb-/- hybridization over the Rb+/+ hybridization. Each bar represents the mean of at least 3 independent experiments (±SE of the mean), with P

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Rb represses the transcription rate of the Pak1 gene. (A) Representative nuclear run-on assay from a triplicate experiment showing hybridization of Rb+/+ ( blot 1 ) and Rb-/- ( blot 2 ) DIG-labeled mRNA probes to a Pak1 cDNA immobilized in a membrane. These two probes were also hybridized to GAPDH cDNAs ( blots 3 and 4 ). Hybridization signal intensity was stronger with the Rb-/- probe, indicating higher transcriptional rate of the Pak1 gene in Rb-/- nuclei. (B) To determine the differences in signal intensity, the chemioluminiscence in the membranes was quantified and analyzed using BioRad’s Quantity 1 Imaging Software and normalized against the signal intensities obtained when hybridizing to the GAPDH cDNAs (blots 3 and 4 in A ). This quantitative analysis showed a statistically significant 2.7-fold increase in the signal intensity of the Rb-/- hybridization over the Rb+/+ hybridization. Each bar represents the mean of at least 3 independent experiments (±SE of the mean), with P

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Nuclear Run-on Assay, Hybridization, Labeling, Imaging, Software

    Rb interacts with the Pak1 promoter in complex with E2F. (A) ChIP assays showing interaction of Rb with the Pak1 promoter. The Pak1 promoter primer set used (-201/+250) is the same as illustrated in Fig 3B . The osteocalcin (OC) promoter was used as a positive control since Rb has been shown to physically interact with this promoter [ 4 ]. Negative controls were performed either by omitting DNA from the PCR reaction (no DNA or ND) or by immunoprecipitating with an irrelevant antibody against GAPDH. L, DNA ladder; In, PCR reactions done with input DNA not subjected to immunoprecipitation; IP, samples immunoprecipitated with anti-Rb antibody. (B) ChIP assays showing that Rb binds to the Pak1 promoter in complex with E2F1. ChIP assays in which the immunoprecipitation was done with anti-E2F1 antibody is labeled as IP-E2F. ChIP assays in which an immunoprecipitation done with an anti-Rb antibody followed by a second round of immunoprecipitation with an anti-E2F1 antibody is labeled as IP-Rb-E2F. PCR using primers against the Pak1 promoter resulted in a product, indicating interaction of the Rb-E2F complex to the Pak1 promoter. L, DNA ladder; In, PCR done with non-immunoprecipitated input DNA; ND, negative control with no DNA added to the PCR reaction.

    Journal: PLoS ONE

    Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

    doi: 10.1371/journal.pone.0142406

    Figure Lengend Snippet: Rb interacts with the Pak1 promoter in complex with E2F. (A) ChIP assays showing interaction of Rb with the Pak1 promoter. The Pak1 promoter primer set used (-201/+250) is the same as illustrated in Fig 3B . The osteocalcin (OC) promoter was used as a positive control since Rb has been shown to physically interact with this promoter [ 4 ]. Negative controls were performed either by omitting DNA from the PCR reaction (no DNA or ND) or by immunoprecipitating with an irrelevant antibody against GAPDH. L, DNA ladder; In, PCR reactions done with input DNA not subjected to immunoprecipitation; IP, samples immunoprecipitated with anti-Rb antibody. (B) ChIP assays showing that Rb binds to the Pak1 promoter in complex with E2F1. ChIP assays in which the immunoprecipitation was done with anti-E2F1 antibody is labeled as IP-E2F. ChIP assays in which an immunoprecipitation done with an anti-Rb antibody followed by a second round of immunoprecipitation with an anti-E2F1 antibody is labeled as IP-Rb-E2F. PCR using primers against the Pak1 promoter resulted in a product, indicating interaction of the Rb-E2F complex to the Pak1 promoter. L, DNA ladder; In, PCR done with non-immunoprecipitated input DNA; ND, negative control with no DNA added to the PCR reaction.

    Article Snippet: For immunoblotting, 50 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose by standard procedures, and immunoblotted with anti-Pak1 (Santa Cruz, sc-882, rabbit polyclonal raised against the N-terminus of Pak1 of rat origin) and anti-α-tubulin (Calbiochem, CP06, mouse monoclonal raised against native chick brain microtubules) primary antibody at 1:100 dilutions.

    Techniques: Chromatin Immunoprecipitation, Positive Control, Polymerase Chain Reaction, Immunoprecipitation, Labeling, Negative Control

    Pak1 promotes PP2A subunit assembly and activation

    Journal: Experimental hematology

    Article Title: A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells

    doi: 10.1016/j.exphem.2012.10.001

    Figure Lengend Snippet: Pak1 promotes PP2A subunit assembly and activation

    Article Snippet: For Pak1 immunoprecipitation, brain was homogenized, sonicated, BCA quantified, and equivalent quantities incubated with anti-Pak1 antibody (Santa Cruz) in protein-A/G-agarose.

    Techniques: Activation Assay

    Pak1 disruption inhibits pT567-ERM dephosphorylation but not cofilin dephosphorylation

    Journal: Experimental hematology

    Article Title: A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells

    doi: 10.1016/j.exphem.2012.10.001

    Figure Lengend Snippet: Pak1 disruption inhibits pT567-ERM dephosphorylation but not cofilin dephosphorylation

    Article Snippet: For Pak1 immunoprecipitation, brain was homogenized, sonicated, BCA quantified, and equivalent quantities incubated with anti-Pak1 antibody (Santa Cruz) in protein-A/G-agarose.

    Techniques: De-Phosphorylation Assay

    Pak1 and PP2A mediate pT567-ERM dephosphorylation and F-actin rearrangement in RBL-2H3 cells

    Journal: Experimental hematology

    Article Title: A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells

    doi: 10.1016/j.exphem.2012.10.001

    Figure Lengend Snippet: Pak1 and PP2A mediate pT567-ERM dephosphorylation and F-actin rearrangement in RBL-2H3 cells

    Article Snippet: For Pak1 immunoprecipitation, brain was homogenized, sonicated, BCA quantified, and equivalent quantities incubated with anti-Pak1 antibody (Santa Cruz) in protein-A/G-agarose.

    Techniques: De-Phosphorylation Assay

    Proposed model for a Pak1-PP2A-ERM axis regulating allergen-induced mast cell degranulation

    Journal: Experimental hematology

    Article Title: A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells

    doi: 10.1016/j.exphem.2012.10.001

    Figure Lengend Snippet: Proposed model for a Pak1-PP2A-ERM axis regulating allergen-induced mast cell degranulation

    Article Snippet: For Pak1 immunoprecipitation, brain was homogenized, sonicated, BCA quantified, and equivalent quantities incubated with anti-Pak1 antibody (Santa Cruz) in protein-A/G-agarose.

    Techniques:

    Pak1 regulates mast cell cytoskeletal rearrangement, degranulation, and systemic histamine release

    Journal: Experimental hematology

    Article Title: A Pak1-PP2A-ERM signaling axis mediates F-actin rearrangement and degranulation in mast cells

    doi: 10.1016/j.exphem.2012.10.001

    Figure Lengend Snippet: Pak1 regulates mast cell cytoskeletal rearrangement, degranulation, and systemic histamine release

    Article Snippet: For Pak1 immunoprecipitation, brain was homogenized, sonicated, BCA quantified, and equivalent quantities incubated with anti-Pak1 antibody (Santa Cruz) in protein-A/G-agarose.

    Techniques:

    JNK, as a downstream effector of PAK1, is essential for MMP-9 but not MMP-2 expression . (A) Normal HDFs, cultured on plastic or in 3D type-I collagen, were treated by cytokines together with inhibitors for p38 MAP kinase, JNK, and PI-3' kinase respectively at their effective concentrations. MMP-9 and -2 in the conditioned medium were detected by zymography. (B) HDFs transduced by lentivirus expressing GFP or PAK1 variant (K299R) were stimulated by cytokines for 20 min. JNK1/2 and the phosphorylated JNK were measured by Western blot analysis. (C) The levels of phosphorylated JNK1/2 were quantitated by densitometry analysis, and normalized by their protein levels. Data are the averages of three experiments.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: JNK, as a downstream effector of PAK1, is essential for MMP-9 but not MMP-2 expression . (A) Normal HDFs, cultured on plastic or in 3D type-I collagen, were treated by cytokines together with inhibitors for p38 MAP kinase, JNK, and PI-3' kinase respectively at their effective concentrations. MMP-9 and -2 in the conditioned medium were detected by zymography. (B) HDFs transduced by lentivirus expressing GFP or PAK1 variant (K299R) were stimulated by cytokines for 20 min. JNK1/2 and the phosphorylated JNK were measured by Western blot analysis. (C) The levels of phosphorylated JNK1/2 were quantitated by densitometry analysis, and normalized by their protein levels. Data are the averages of three experiments.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Cell Culture, Zymography, Variant Assay, Western Blot

    Activation and stabilization of endogenous PAK1 in response to TNF-α by keratinocytes and dermal fibroblast . (A) Keratinocytes (IKCs) were treated by TNF-α for the indicated periods and PAK1 protein was measured by Western blot analysis. Intact PAK1 and the putative degradation and non-specific bands were indicated. (B) Threonine-423 phosphorylation of PAK1 by the IKCs in response to TNF-α was measured by Western blot analysis. (C) Threonine-212 phosphorylation of PAK1 by the IKCs in response to TNF-α was measured by WB analysis. (D) Distribution of phosphorylated PAK1 in cytosolic and membrane compartments was measured by Western blot analysis. GAPDH was used as an indicator for cytosolic proteins. (E) Threonine-423 phosphorylation of PAK1 by HDFs was measured by Western blot analysis. (F) Thronine-212 phosphorylation by HDFs was measured by WB analysis.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: Activation and stabilization of endogenous PAK1 in response to TNF-α by keratinocytes and dermal fibroblast . (A) Keratinocytes (IKCs) were treated by TNF-α for the indicated periods and PAK1 protein was measured by Western blot analysis. Intact PAK1 and the putative degradation and non-specific bands were indicated. (B) Threonine-423 phosphorylation of PAK1 by the IKCs in response to TNF-α was measured by Western blot analysis. (C) Threonine-212 phosphorylation of PAK1 by the IKCs in response to TNF-α was measured by WB analysis. (D) Distribution of phosphorylated PAK1 in cytosolic and membrane compartments was measured by Western blot analysis. GAPDH was used as an indicator for cytosolic proteins. (E) Threonine-423 phosphorylation of PAK1 by HDFs was measured by Western blot analysis. (F) Thronine-212 phosphorylation by HDFs was measured by WB analysis.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Activation Assay, Western Blot

    NF-κB pathway is required for MMP-9 expression, but is independent of PAK1 and p38 MAP kinase . (A) HDFs transduced by lentivrius expressing GFP, PAK1 (K299R), p38 MAP kinase, and its dominant negative ones were cultured on plastic and treated by TNF-α for the indicated intervals. Degradation of I-kappaB was measured by Western blot analysis. (B) HDFs were treated by TNF-α with IKK inhibitors at the indicated concentration for 6 hrs. The mRNA of MMP-9 was measured by real-time RT-PCR analysis. Results are from the average of three experiments. (C) HDFs were treated by TNF-α with IKK inhibitors at low concentration for 3 days. MMP-9 and -2 were measured by zymography.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: NF-κB pathway is required for MMP-9 expression, but is independent of PAK1 and p38 MAP kinase . (A) HDFs transduced by lentivrius expressing GFP, PAK1 (K299R), p38 MAP kinase, and its dominant negative ones were cultured on plastic and treated by TNF-α for the indicated intervals. Degradation of I-kappaB was measured by Western blot analysis. (B) HDFs were treated by TNF-α with IKK inhibitors at the indicated concentration for 6 hrs. The mRNA of MMP-9 was measured by real-time RT-PCR analysis. Results are from the average of three experiments. (C) HDFs were treated by TNF-α with IKK inhibitors at low concentration for 3 days. MMP-9 and -2 were measured by zymography.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Dominant Negative Mutation, Cell Culture, Western Blot, Concentration Assay, Quantitative RT-PCR, Zymography

    IL-1 stimulation of PAK1 phosphorylation by primary rat hepatic stellate cells . Primary rat hepatic stellate cells were stimulated by IL-1α for the indicated time. Phosphorylation of PAK1 at threonine 423 and threonine-212 and GAPDH were measured by Western blot analysis.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: IL-1 stimulation of PAK1 phosphorylation by primary rat hepatic stellate cells . Primary rat hepatic stellate cells were stimulated by IL-1α for the indicated time. Phosphorylation of PAK1 at threonine 423 and threonine-212 and GAPDH were measured by Western blot analysis.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Western Blot

    PAK1 mediates TNF-α-induced MMP-9 but not MMP-2 . (A) HDFs and the cells transduced by lentivrius expressing GFP or PAK1 variants (K299R, catalytic inactive) were cultured in 3D type-I collagen. After culture for 3 days with TNF-α at the indicated concentrations, the gelatinases in the conditioned medium were measured by zymography. (B) HDFs expressing GFP or a PAK1 variant were cultured on plastic or in 3D type-I collagen and treated by TNF-α or/and TGF-β for 16 hrs. The mRNA of MMP-9 was measured by real-time RT-PCR and normalized by GAPDH. The results are representative of three independent experiments. (C) The activities of 670-bp 5'-promoter of human MMP-9 were measured by transient transfection of HDFs expressing GFP or PAK1 mutant (K299R) with pGL2 reporter plasmids encoding firefly luciferase and CMV promoter driving renilla luciferase. The resultant cells were stimulated by cytokines for 16 hrs (n = 6). (D) HDFs ectopically expressed PAK1 protein were stimulated by TNF-α for 3 days. PAK1 protein was measured by Western blot analysis. Of note is the increased the viral promoter driven PAK1 protein in response to TNF-α. (E) HDFs expressing PAK1 were treated with or without TNF-α together with cycloheximide (20 μg/ml). At the indicated intervals the cells were harvested for Western blot analysis of PAK1.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: PAK1 mediates TNF-α-induced MMP-9 but not MMP-2 . (A) HDFs and the cells transduced by lentivrius expressing GFP or PAK1 variants (K299R, catalytic inactive) were cultured in 3D type-I collagen. After culture for 3 days with TNF-α at the indicated concentrations, the gelatinases in the conditioned medium were measured by zymography. (B) HDFs expressing GFP or a PAK1 variant were cultured on plastic or in 3D type-I collagen and treated by TNF-α or/and TGF-β for 16 hrs. The mRNA of MMP-9 was measured by real-time RT-PCR and normalized by GAPDH. The results are representative of three independent experiments. (C) The activities of 670-bp 5'-promoter of human MMP-9 were measured by transient transfection of HDFs expressing GFP or PAK1 mutant (K299R) with pGL2 reporter plasmids encoding firefly luciferase and CMV promoter driving renilla luciferase. The resultant cells were stimulated by cytokines for 16 hrs (n = 6). (D) HDFs ectopically expressed PAK1 protein were stimulated by TNF-α for 3 days. PAK1 protein was measured by Western blot analysis. Of note is the increased the viral promoter driven PAK1 protein in response to TNF-α. (E) HDFs expressing PAK1 were treated with or without TNF-α together with cycloheximide (20 μg/ml). At the indicated intervals the cells were harvested for Western blot analysis of PAK1.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Cell Culture, Zymography, Variant Assay, Quantitative RT-PCR, Transfection, Mutagenesis, Luciferase, Western Blot

    TNF-α-induced expression of MMP-9 through PAK1, and stabilization of the ectopically expressed kinase by human keratinocytes . Immortalized human keratinocytes transduced by lentivrius expressing GFP or PAK1 variants were cultured in KGM. After culture for 3 days with cytokines the gelatinases in the conditioned medium were measured by zymography. (B) PAK1 and a non-specific protein as a negative control were measured by Western blot analysis.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: TNF-α-induced expression of MMP-9 through PAK1, and stabilization of the ectopically expressed kinase by human keratinocytes . Immortalized human keratinocytes transduced by lentivrius expressing GFP or PAK1 variants were cultured in KGM. After culture for 3 days with cytokines the gelatinases in the conditioned medium were measured by zymography. (B) PAK1 and a non-specific protein as a negative control were measured by Western blot analysis.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Cell Culture, Zymography, Negative Control, Western Blot

    MMP-9, but not other MMPs, is specifically regulated by PAK1 . (A) HDFs transduced by lentivrus expressing GFP or PAK1 variant (K299R) were embedded in type-I collagen and treated by cytokines for 3 days. MMP-3 in the conditioned medium was measured by Western blot analysis. (B) After 16 hrs of treatment with cytokines, the mRNA of MMPs and TIMP-1 were measured by real-time RT-PCR, normalized by GAPDH (n = 3).

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: MMP-9, but not other MMPs, is specifically regulated by PAK1 . (A) HDFs transduced by lentivrus expressing GFP or PAK1 variant (K299R) were embedded in type-I collagen and treated by cytokines for 3 days. MMP-3 in the conditioned medium was measured by Western blot analysis. (B) After 16 hrs of treatment with cytokines, the mRNA of MMPs and TIMP-1 were measured by real-time RT-PCR, normalized by GAPDH (n = 3).

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Variant Assay, Western Blot, Quantitative RT-PCR

    p38 MAP kinase is not required for MMP-9 expression and stability of the kinase is not regulated by TNF-α . (A) HDFs transduced by lentivirus expressing GFP, PAK1 (K299R), p38 MAP kinase, and its dominant negative one were cultured on plastic or in 3D type-I collagen, followed by stimulation by cytokines. After 3 days, MMP-9 and -2 in the conditioned medium were measured by zymography. (B) Protein of p38MAP kinase was measured by Western blot analysis.

    Journal: BMC Immunology

    Article Title: Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    doi: 10.1186/1471-2172-10-15

    Figure Lengend Snippet: p38 MAP kinase is not required for MMP-9 expression and stability of the kinase is not regulated by TNF-α . (A) HDFs transduced by lentivirus expressing GFP, PAK1 (K299R), p38 MAP kinase, and its dominant negative one were cultured on plastic or in 3D type-I collagen, followed by stimulation by cytokines. After 3 days, MMP-9 and -2 in the conditioned medium were measured by zymography. (B) Protein of p38MAP kinase was measured by Western blot analysis.

    Article Snippet: Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies.

    Techniques: Expressing, Dominant Negative Mutation, Cell Culture, Zymography, Western Blot

    Disruption of Pak1 sensitizes the myocardium to I/R-induced ventricular arrhythmias.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury

    doi: 10.1152/ajpheart.01003.2010

    Figure Lengend Snippet: Disruption of Pak1 sensitizes the myocardium to I/R-induced ventricular arrhythmias.

    Article Snippet: Rabbit anti-Pak1, rabbit anti-phospho-Pak1 Thr423, rabbit anti-Akt, and anti-phospho-Akt Thr308 residue, were all purchased from Cell Signaling Technology (Hitchin, UK).

    Techniques:

    FTY720 induces Pak1 and Akt activation. A and B : representative blots for activation of phospho-Akt and phospho-Pak1 in isolated neonatal rat myocytes treated with FTY720. Myocytes were exposed for 15 min to 25 nM FTY720. C and D : representative blots

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury

    doi: 10.1152/ajpheart.01003.2010

    Figure Lengend Snippet: FTY720 induces Pak1 and Akt activation. A and B : representative blots for activation of phospho-Akt and phospho-Pak1 in isolated neonatal rat myocytes treated with FTY720. Myocytes were exposed for 15 min to 25 nM FTY720. C and D : representative blots

    Article Snippet: Rabbit anti-Pak1, rabbit anti-phospho-Pak1 Thr423, rabbit anti-Akt, and anti-phospho-Akt Thr308 residue, were all purchased from Cell Signaling Technology (Hitchin, UK).

    Techniques: Activation Assay, Isolation

    PAK1 overexpression exerts anti‐atrophic effects in two models of in vitro C2C12 atrophy. (A) The circulating levels of IL6 in the plasma of C26‐bearing mice is drastically increased with respect to PBS‐injected mice ( n = 3–6, Mann Whitney's test, * P = 0.02). (B) Upon 5 h treatment with 10 or 100 ng/mL murine IL6, C2C12 myoblasts transiently expressing Stat3 4X‐FLuc reporter plasmids induce FLuc ( n = 3, one‐way anova followed by Dunnett's test, ** P ≤ 0.01, **** P ≤ 0.0001). (C) IL6‐induced Stat3‐FLuc signal is reduced in myoblasts expressing GFP‐PAK1 with respect to those expressing GFP. Myoblasts treated for 5 h with IL6 (100 ng/mL) were previously transfected with Stat3 4X‐FLuc plasmid, Renilla luciferase plasmids, and GFP or PAK1‐expressing plasmids. The results of three independent experiments are shown. Mean is reported ( n = 14, unpaired t ‐test, *** P = 0.0002). (D) Rates of long‐lived protein degradation were measured in myotubes transfected on the third day of differentiation with plasmids for pDSRed2‐PAK1 or empty vector and differentiated for one more day, when they were exposed for 24 h to 10 ng/mL IL6 ( n = 4, unpaired t ‐test, * P ≤ 0.05). (E) The mRNA content of Pak1 is reduced in atrophying myotubes exposed for 24 h to 10 μM dexamethasone ( n = 6, Mann Whitney's test, ** P = 0.002, **** P ≤ 0.0001). (F) Myotubes transfected on the third day of differentiation with plasmids for GFP‐PAK1 or only GFP were exposed for 24 h on the fourth day of differentiation to vehicle or 1 μM dexamethasone or 10 μM IPA‐3. In these conditions, the mRNA levels of MuRF1 were determined by quantitative polymerase chain reaction ( n = 4, unpaired t ‐test, * P ≤ 0.05). (G) Myoblasts were transfected, as indicated earlier, for 24 h, total protein was then extracted. Immunoblot analysis reveals the protein content of p‐FOXO3, FOXO3, atrogin‐1, and vinculin that is used as loading control. (H) Quantification of the ratio between p‐FOXO3 over total FOXO3 is shown ( n = 6–8, unpaired t ‐test, * P ≤ 0.05). SEM is indicated in all figures. IL6, interleukin‐6; FLuc, firefly luciferase; PAK1, p21 protein‐activated kinase 1; TBP, Tata‐Binding Protein.

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy) Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy

    doi: 10.1002/jcsm.12303

    Figure Lengend Snippet: PAK1 overexpression exerts anti‐atrophic effects in two models of in vitro C2C12 atrophy. (A) The circulating levels of IL6 in the plasma of C26‐bearing mice is drastically increased with respect to PBS‐injected mice ( n = 3–6, Mann Whitney's test, * P = 0.02). (B) Upon 5 h treatment with 10 or 100 ng/mL murine IL6, C2C12 myoblasts transiently expressing Stat3 4X‐FLuc reporter plasmids induce FLuc ( n = 3, one‐way anova followed by Dunnett's test, ** P ≤ 0.01, **** P ≤ 0.0001). (C) IL6‐induced Stat3‐FLuc signal is reduced in myoblasts expressing GFP‐PAK1 with respect to those expressing GFP. Myoblasts treated for 5 h with IL6 (100 ng/mL) were previously transfected with Stat3 4X‐FLuc plasmid, Renilla luciferase plasmids, and GFP or PAK1‐expressing plasmids. The results of three independent experiments are shown. Mean is reported ( n = 14, unpaired t ‐test, *** P = 0.0002). (D) Rates of long‐lived protein degradation were measured in myotubes transfected on the third day of differentiation with plasmids for pDSRed2‐PAK1 or empty vector and differentiated for one more day, when they were exposed for 24 h to 10 ng/mL IL6 ( n = 4, unpaired t ‐test, * P ≤ 0.05). (E) The mRNA content of Pak1 is reduced in atrophying myotubes exposed for 24 h to 10 μM dexamethasone ( n = 6, Mann Whitney's test, ** P = 0.002, **** P ≤ 0.0001). (F) Myotubes transfected on the third day of differentiation with plasmids for GFP‐PAK1 or only GFP were exposed for 24 h on the fourth day of differentiation to vehicle or 1 μM dexamethasone or 10 μM IPA‐3. In these conditions, the mRNA levels of MuRF1 were determined by quantitative polymerase chain reaction ( n = 4, unpaired t ‐test, * P ≤ 0.05). (G) Myoblasts were transfected, as indicated earlier, for 24 h, total protein was then extracted. Immunoblot analysis reveals the protein content of p‐FOXO3, FOXO3, atrogin‐1, and vinculin that is used as loading control. (H) Quantification of the ratio between p‐FOXO3 over total FOXO3 is shown ( n = 6–8, unpaired t ‐test, * P ≤ 0.05). SEM is indicated in all figures. IL6, interleukin‐6; FLuc, firefly luciferase; PAK1, p21 protein‐activated kinase 1; TBP, Tata‐Binding Protein.

    Article Snippet: The antibodies used were as follows: rabbit anti‐Pak1 (1:1000, Cell Signalling 2602), rabbit anti‐Pak1/2/3 (1:1000, Cell Signalling 2604), rabbit anti‐phospho‐PAK1 (Ser144)/PAK2 (Ser141) (1:1000, Cell Signalling 2606), rabbit anti‐phospho‐PAK1 (Thr423)/PAK2 (Thr402) (1:1000, Cell Signalling 2601), rabbit anti‐FoxO3a (1:1000, Cell Signalling 2497), rabbit anti‐phospho‐FoxO3a (Ser253) (1:1000, Cell Signalling 9466), mouse anti‐atrogin1 (1:1000, Abcam ab168372), rabbit anti‐phospho p38 (1:1000, Cell Signalling 9211), rabbit anti‐p38 (1:1000, Cell Signalling 9212), mouse anti‐myogenin (1:500, e‐Bioscience 14‐5643), rabbit anti‐tubulin antibody (1:500, Santa Cruz sc‐9104), rabbit anti‐actin (1:1000, Sigma A2066), mouse anti‐vinculin (1:1000, Abcam ab18058), and mouse anti‐vinculin (1:5000, Sigma, V9264).

    Techniques: Over Expression, In Vitro, Mouse Assay, Injection, MANN-WHITNEY, Expressing, Transfection, Plasmid Preparation, Luciferase, Indirect Immunoperoxidase Assay, Real-time Polymerase Chain Reaction, Binding Assay

    IPA‐3 treatment reduces myogenin expression throughout Mabs and C2C12 myogenic differentiation process. (A) Immunoblot analysis revealing p‐Pak1 (S144) and total Pak1 in C2C12 protein extracts following 2 h treatment with various concentrations of the inhibitor IPA‐3 or vehicle (dimethyl sulfoxide). Pak1 and p‐Pak1 signals have been developed on separate filters and vinculin serves as a loading control. (B) Immunofluorescence microphotographs of Mabs labelled with an antibody against myogenin (green). (C, D) Bar plot representing the percentage of myogenin positive cells for Mabs and C2C12, respectively. (E) The graph illustrates the percentage difference of myogenin positive cells during Mabs myogenic differentiation program. (F) Graph showing the different percentage of myogenin positive cells during C2C12 differentiation. The values represented are means of at least three independent experiments + SEM. Statistical significance has been evaluated using the unpaired t ‐test, * P ≤ 0.05, ** P ≤ 0.01. Scale bar: 100 μm. PAK1, p21 protein‐activated kinase 1; Mabs, mesoangioblasts.

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy) Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy

    doi: 10.1002/jcsm.12303

    Figure Lengend Snippet: IPA‐3 treatment reduces myogenin expression throughout Mabs and C2C12 myogenic differentiation process. (A) Immunoblot analysis revealing p‐Pak1 (S144) and total Pak1 in C2C12 protein extracts following 2 h treatment with various concentrations of the inhibitor IPA‐3 or vehicle (dimethyl sulfoxide). Pak1 and p‐Pak1 signals have been developed on separate filters and vinculin serves as a loading control. (B) Immunofluorescence microphotographs of Mabs labelled with an antibody against myogenin (green). (C, D) Bar plot representing the percentage of myogenin positive cells for Mabs and C2C12, respectively. (E) The graph illustrates the percentage difference of myogenin positive cells during Mabs myogenic differentiation program. (F) Graph showing the different percentage of myogenin positive cells during C2C12 differentiation. The values represented are means of at least three independent experiments + SEM. Statistical significance has been evaluated using the unpaired t ‐test, * P ≤ 0.05, ** P ≤ 0.01. Scale bar: 100 μm. PAK1, p21 protein‐activated kinase 1; Mabs, mesoangioblasts.

    Article Snippet: The antibodies used were as follows: rabbit anti‐Pak1 (1:1000, Cell Signalling 2602), rabbit anti‐Pak1/2/3 (1:1000, Cell Signalling 2604), rabbit anti‐phospho‐PAK1 (Ser144)/PAK2 (Ser141) (1:1000, Cell Signalling 2606), rabbit anti‐phospho‐PAK1 (Thr423)/PAK2 (Thr402) (1:1000, Cell Signalling 2601), rabbit anti‐FoxO3a (1:1000, Cell Signalling 2497), rabbit anti‐phospho‐FoxO3a (Ser253) (1:1000, Cell Signalling 9466), mouse anti‐atrogin1 (1:1000, Abcam ab168372), rabbit anti‐phospho p38 (1:1000, Cell Signalling 9211), rabbit anti‐p38 (1:1000, Cell Signalling 9212), mouse anti‐myogenin (1:500, e‐Bioscience 14‐5643), rabbit anti‐tubulin antibody (1:500, Santa Cruz sc‐9104), rabbit anti‐actin (1:1000, Sigma A2066), mouse anti‐vinculin (1:1000, Abcam ab18058), and mouse anti‐vinculin (1:5000, Sigma, V9264).

    Techniques: Indirect Immunoperoxidase Assay, Expressing, Immunofluorescence

    Pak1 expression is reduced in TA muscles of cachectic C26‐bearing mice and its ectopic expression preserves myofiber area of cachectic mice by reducing the expression of atrogin‐1 and MuRF1 and possibly by inducing myogenin . (A) Representative western blot revealing total Pak1 in crude protein extracts from TA of colon adenocarcinoma‐bearing mice (C26) compared with controls (PBS). Vinculin is used as a loading control. Twenty microgram of lysates of C2C12 myoblasts previously transfected for 24 h with GFP‐PAK1 expressing plasmids have been used as controls as well as non‐transfected cells. (B) The bar graph illustrates the densitometric quantification of Pak1/vinculin signal ratio for experiments as represented in (A) ( n = 4–6, unpaired t ‐ test, ** P ≤ 0.01) GUSB has been used as housekeeping gene. (C) The mRNA levels of Pak1 in TA from C26‐bearing mice were determined by quantitative polymerase chain reaction ( n = 6–10, unpaired t ‐test, * P = 0.02). (D) Representative images of cross‐sections of TA from C26‐bearing mice or PBS‐injected ones, previously electroporated in vivo with DsRed2‐PAK1, are shown. Scale bar: 100 μm. (E) Frequency histograms showing the distribution of cross‐sectional areas of muscle fibers of TA either from PBS‐injected mice or C26‐injected ones for 14 days and transfected with empty vector or DsRed2‐PAK1. (F) The mean cross‐sectional area is shown for the four conditions described earlier ( n = 219 fibers from five electroporated TA from PBS‐injected mice; n = 339 fibers from seven electroporated TA from C26‐injected mice, unpaired t ‐test, **** P ≤ 0.001). Atrogin‐1 (G) and MuRF1 (H) expressions (reported in AU) inversely correlate with transfected human GFP‐PAK1 in muscle from C26‐bearing mice. On the x and y axes, the relative amount of expression of the genes indicated is reported. These numbers have been obtained by comparing the CT of the samples with that of standards of which serial dilutions have been run and probed for the same genes in the same plate. Pearson's test is shown for correlation analysis, n = 6–7. (I) The mRNA levels of myogenin in TA from C26‐bearing mice electroporated with plasmids for GFP or GFP‐PAK1 were determined by quantitative polymerase chain reaction ( n = 8–9, unpaired t ‐test, * P ≤ 0.05) TBP has been used as housekeeping gene. SEM is indicated in all figures. PAK1, p21 protein‐activated kinase 1; TA, tibialis anterior; AU, arbitrary units; GUSB, β‐glucuronidase; TBP, Tata‐Binding Protein.

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy) Group I Paks support muscle regeneration and counteract cancer‐associated muscle atrophy

    doi: 10.1002/jcsm.12303

    Figure Lengend Snippet: Pak1 expression is reduced in TA muscles of cachectic C26‐bearing mice and its ectopic expression preserves myofiber area of cachectic mice by reducing the expression of atrogin‐1 and MuRF1 and possibly by inducing myogenin . (A) Representative western blot revealing total Pak1 in crude protein extracts from TA of colon adenocarcinoma‐bearing mice (C26) compared with controls (PBS). Vinculin is used as a loading control. Twenty microgram of lysates of C2C12 myoblasts previously transfected for 24 h with GFP‐PAK1 expressing plasmids have been used as controls as well as non‐transfected cells. (B) The bar graph illustrates the densitometric quantification of Pak1/vinculin signal ratio for experiments as represented in (A) ( n = 4–6, unpaired t ‐ test, ** P ≤ 0.01) GUSB has been used as housekeeping gene. (C) The mRNA levels of Pak1 in TA from C26‐bearing mice were determined by quantitative polymerase chain reaction ( n = 6–10, unpaired t ‐test, * P = 0.02). (D) Representative images of cross‐sections of TA from C26‐bearing mice or PBS‐injected ones, previously electroporated in vivo with DsRed2‐PAK1, are shown. Scale bar: 100 μm. (E) Frequency histograms showing the distribution of cross‐sectional areas of muscle fibers of TA either from PBS‐injected mice or C26‐injected ones for 14 days and transfected with empty vector or DsRed2‐PAK1. (F) The mean cross‐sectional area is shown for the four conditions described earlier ( n = 219 fibers from five electroporated TA from PBS‐injected mice; n = 339 fibers from seven electroporated TA from C26‐injected mice, unpaired t ‐test, **** P ≤ 0.001). Atrogin‐1 (G) and MuRF1 (H) expressions (reported in AU) inversely correlate with transfected human GFP‐PAK1 in muscle from C26‐bearing mice. On the x and y axes, the relative amount of expression of the genes indicated is reported. These numbers have been obtained by comparing the CT of the samples with that of standards of which serial dilutions have been run and probed for the same genes in the same plate. Pearson's test is shown for correlation analysis, n = 6–7. (I) The mRNA levels of myogenin in TA from C26‐bearing mice electroporated with plasmids for GFP or GFP‐PAK1 were determined by quantitative polymerase chain reaction ( n = 8–9, unpaired t ‐test, * P ≤ 0.05) TBP has been used as housekeeping gene. SEM is indicated in all figures. PAK1, p21 protein‐activated kinase 1; TA, tibialis anterior; AU, arbitrary units; GUSB, β‐glucuronidase; TBP, Tata‐Binding Protein.

    Article Snippet: The antibodies used were as follows: rabbit anti‐Pak1 (1:1000, Cell Signalling 2602), rabbit anti‐Pak1/2/3 (1:1000, Cell Signalling 2604), rabbit anti‐phospho‐PAK1 (Ser144)/PAK2 (Ser141) (1:1000, Cell Signalling 2606), rabbit anti‐phospho‐PAK1 (Thr423)/PAK2 (Thr402) (1:1000, Cell Signalling 2601), rabbit anti‐FoxO3a (1:1000, Cell Signalling 2497), rabbit anti‐phospho‐FoxO3a (Ser253) (1:1000, Cell Signalling 9466), mouse anti‐atrogin1 (1:1000, Abcam ab168372), rabbit anti‐phospho p38 (1:1000, Cell Signalling 9211), rabbit anti‐p38 (1:1000, Cell Signalling 9212), mouse anti‐myogenin (1:500, e‐Bioscience 14‐5643), rabbit anti‐tubulin antibody (1:500, Santa Cruz sc‐9104), rabbit anti‐actin (1:1000, Sigma A2066), mouse anti‐vinculin (1:1000, Abcam ab18058), and mouse anti‐vinculin (1:5000, Sigma, V9264).

    Techniques: Expressing, Mouse Assay, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Injection, In Vivo, Plasmid Preparation, Binding Assay

    Dominant-negative PID expression inhibits cell viability and PAK phosphorylation in human MM cell lines. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3 and β-actin levels 72-hr post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22

    Journal: Molecular cancer research : MCR

    Article Title: Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival Through the AKT1 and Raf-MAPK Pathways

    doi: 10.1158/1541-7786.MCR-12-0082

    Figure Lengend Snippet: Dominant-negative PID expression inhibits cell viability and PAK phosphorylation in human MM cell lines. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3 and β-actin levels 72-hr post-nucleofection of pcDNA or pcDNA-PID in Me12 and Meso 22

    Article Snippet: Antibodies specific for total PAK1 and PAK1/2/3 (Cell Signaling) were used to probe the membrane and determine where specific PAK isoforms ran on an SDS-PAGE gel.

    Techniques: Dominant Negative Mutation, Expressing

    Identification of AKT and MAPK signaling as potentially important effector pathways in regulated tumor cell viability. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3, P-AKT, total AKT, P-ERK1/2, and β-actin in Me12 and Meso 22 treated with

    Journal: Molecular cancer research : MCR

    Article Title: Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival Through the AKT1 and Raf-MAPK Pathways

    doi: 10.1158/1541-7786.MCR-12-0082

    Figure Lengend Snippet: Identification of AKT and MAPK signaling as potentially important effector pathways in regulated tumor cell viability. A) Immunoblot analysis of P-PAK1/2/3, total PAK1/2/3, P-AKT, total AKT, P-ERK1/2, and β-actin in Me12 and Meso 22 treated with

    Article Snippet: Antibodies specific for total PAK1 and PAK1/2/3 (Cell Signaling) were used to probe the membrane and determine where specific PAK isoforms ran on an SDS-PAGE gel.

    Techniques:

    PAKs are activated in MM specimens and cell lines. A) Immunohistochemical-staining of human MM and normal lung pleura with a phospho-specific antibody against Ser141 of PAK1/2/3 (P-PAK1/2/3). B) Immunohistochemical staining of asbestos-induced MMs from

    Journal: Molecular cancer research : MCR

    Article Title: Group I p21-Activated Kinases (PAKs) Promote Tumor Cell Proliferation and Survival Through the AKT1 and Raf-MAPK Pathways

    doi: 10.1158/1541-7786.MCR-12-0082

    Figure Lengend Snippet: PAKs are activated in MM specimens and cell lines. A) Immunohistochemical-staining of human MM and normal lung pleura with a phospho-specific antibody against Ser141 of PAK1/2/3 (P-PAK1/2/3). B) Immunohistochemical staining of asbestos-induced MMs from

    Article Snippet: Antibodies specific for total PAK1 and PAK1/2/3 (Cell Signaling) were used to probe the membrane and determine where specific PAK isoforms ran on an SDS-PAGE gel.

    Techniques: Immunohistochemistry, Staining

    Nischarin interacts with PAK1/2 and regulates PAK activity. Lysates (500 μl, concentration 3 μg/μl) of rat cortical neurons were incubated with an anti-rabbit PAK1 polyclonal antibody, an anti-rabbit PAK2 polyclonal antibody or an anti-mouse Nischarin monoclonal antibody and their IgG then precipitated with G-Sepharose protein beads. (A) The Nischarin immunoprecipitates were blotted with anti-PAK1 antibody (top panel) or with anti-PAK2 antibody (bottom panel). (B) PAK1 or PAK2 immunoprecipitates were blotted with anti-Nischarin antibody. IgG was used as the negative control and 5% cell lysates (input) was added in the gel in each experiment to be a positive control. IP EX: immunoprecipitation extract. (C) Immunofluorescence staining was performed on primary cultured hippocampal neurons (10 DIV). Strong staining for Nischarin (green) co-localized both with PAK1 (red, left panel) and PAK2 (red, right panel) in the perinuclear region and the dendrites (arrows). Scale bars: 10 μm. (D, E) Nischarin-shRNA effectively suppresses the expression of Nischarin protein and stimulates the phosphorylation of both PAK1 and PAK2, in Neuro-2a cells (n = 3). Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (* p

    Journal: PLoS ONE

    Article Title: Inhibition of Nischarin Expression Promotes Neurite Outgrowth through Regulation of PAK Activity

    doi: 10.1371/journal.pone.0144948

    Figure Lengend Snippet: Nischarin interacts with PAK1/2 and regulates PAK activity. Lysates (500 μl, concentration 3 μg/μl) of rat cortical neurons were incubated with an anti-rabbit PAK1 polyclonal antibody, an anti-rabbit PAK2 polyclonal antibody or an anti-mouse Nischarin monoclonal antibody and their IgG then precipitated with G-Sepharose protein beads. (A) The Nischarin immunoprecipitates were blotted with anti-PAK1 antibody (top panel) or with anti-PAK2 antibody (bottom panel). (B) PAK1 or PAK2 immunoprecipitates were blotted with anti-Nischarin antibody. IgG was used as the negative control and 5% cell lysates (input) was added in the gel in each experiment to be a positive control. IP EX: immunoprecipitation extract. (C) Immunofluorescence staining was performed on primary cultured hippocampal neurons (10 DIV). Strong staining for Nischarin (green) co-localized both with PAK1 (red, left panel) and PAK2 (red, right panel) in the perinuclear region and the dendrites (arrows). Scale bars: 10 μm. (D, E) Nischarin-shRNA effectively suppresses the expression of Nischarin protein and stimulates the phosphorylation of both PAK1 and PAK2, in Neuro-2a cells (n = 3). Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (* p

    Article Snippet: After 2 h of blocking in 5% nonfat milk, blots were incubated overnight at 4°C with the following primary antibodies: anti-Nischarin (1:1000, BD Biosciences, San Jose, USA), anti-PAK1 and anti-PAK2 (1:500, Abcam, Cambridge, MA, USA), anti- phospho-PAK1 (Thr423)/PAK2 (Thr402)(1:1000, Cell Signaling Technology, Beverly, MA, USA) and anti-GAPDH (1:500, Good Here, Hangzhou, China).

    Techniques: Activity Assay, Concentration Assay, Incubation, Negative Control, Positive Control, Immunoprecipitation, Immunofluorescence, Staining, Cell Culture, shRNA, Expressing, CTL Assay

    PAK1 activity is important for Nischarin-regulated neurite outgrowth in Neuro-2a cells. (A) Representative images of Neuro-2a cells transfected with Nis-shRNA-3 and treated with the PAK1 inhibitor IPA3. Scale bar: 20 μm. (B) Neuro-2a cells were transfected with Nis-shRNA-3 and treated with IPA3. Phosphorylation of PAK1/2 was determined by western blot assay. IPA3 treatment reversed the increases in the number of neurite-bearing cells (C) , the mean length of the longest neurite (D) , and total length of neurite per cell (E) in Nischarin-suppressed cells. Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (*** p

    Journal: PLoS ONE

    Article Title: Inhibition of Nischarin Expression Promotes Neurite Outgrowth through Regulation of PAK Activity

    doi: 10.1371/journal.pone.0144948

    Figure Lengend Snippet: PAK1 activity is important for Nischarin-regulated neurite outgrowth in Neuro-2a cells. (A) Representative images of Neuro-2a cells transfected with Nis-shRNA-3 and treated with the PAK1 inhibitor IPA3. Scale bar: 20 μm. (B) Neuro-2a cells were transfected with Nis-shRNA-3 and treated with IPA3. Phosphorylation of PAK1/2 was determined by western blot assay. IPA3 treatment reversed the increases in the number of neurite-bearing cells (C) , the mean length of the longest neurite (D) , and total length of neurite per cell (E) in Nischarin-suppressed cells. Asterisks indicate significant differences between ctl-shRNA and Nis-shRNA (*** p

    Article Snippet: After 2 h of blocking in 5% nonfat milk, blots were incubated overnight at 4°C with the following primary antibodies: anti-Nischarin (1:1000, BD Biosciences, San Jose, USA), anti-PAK1 and anti-PAK2 (1:500, Abcam, Cambridge, MA, USA), anti- phospho-PAK1 (Thr423)/PAK2 (Thr402)(1:1000, Cell Signaling Technology, Beverly, MA, USA) and anti-GAPDH (1:500, Good Here, Hangzhou, China).

    Techniques: Activity Assay, Transfection, shRNA, Western Blot, CTL Assay

    Group-I PAKs induce direct phosphorylation of HACE1. ( a ) In vitro [γ 32 P]-ATP kinase assay using recombinant 6His-HACE1 and recombinant 6His-PAK1 analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. ( b ) MCF12A and HUVEC protein lysates analyzed by immunoblot (IB) using anti-PAK1 and anti-PAK2 antibodies. IB: actin is used as a loading control. ( c–e ) Protein lysates from ( c ) HUVECs and ( d,e ) MCF12A cells transfected with siRNAs targeting ( c , d ) PAK2 or ( e ) PAK1 and plasmid expressing HA-HACE1 and myc-Rac1(Q61L) and analyzed by IB using the indicated antibodies. ( f ) Protein lysates from MCF12A cells transfected with HA-HACE1, either intoxicated with CNF1 for 16 hours or co-transfected with myc-Rac1(Q61L), and treated with FRAX597 at the indicated concentration for 16 hours before IB analysis. ( g , h ) Protein lysates from MCF12A cells transfected with HA-HACE1, myc-Rac1(Q61L) and GST-KID2 or Flag-PAK1K141A and analyzed by IB. IBs in ( a – h .

    Journal: Scientific Reports

    Article Title: Group-I PAKs-mediated phosphorylation of HACE1 at serine 385 regulates its oligomerization state and Rac1 ubiquitination

    doi: 10.1038/s41598-018-19471-2

    Figure Lengend Snippet: Group-I PAKs induce direct phosphorylation of HACE1. ( a ) In vitro [γ 32 P]-ATP kinase assay using recombinant 6His-HACE1 and recombinant 6His-PAK1 analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. ( b ) MCF12A and HUVEC protein lysates analyzed by immunoblot (IB) using anti-PAK1 and anti-PAK2 antibodies. IB: actin is used as a loading control. ( c–e ) Protein lysates from ( c ) HUVECs and ( d,e ) MCF12A cells transfected with siRNAs targeting ( c , d ) PAK2 or ( e ) PAK1 and plasmid expressing HA-HACE1 and myc-Rac1(Q61L) and analyzed by IB using the indicated antibodies. ( f ) Protein lysates from MCF12A cells transfected with HA-HACE1, either intoxicated with CNF1 for 16 hours or co-transfected with myc-Rac1(Q61L), and treated with FRAX597 at the indicated concentration for 16 hours before IB analysis. ( g , h ) Protein lysates from MCF12A cells transfected with HA-HACE1, myc-Rac1(Q61L) and GST-KID2 or Flag-PAK1K141A and analyzed by IB. IBs in ( a – h .

    Article Snippet: SMART-pool siRNA mixes against PAK1 (#5058) and PAK2 (#5062) were acquired from GE Dharmacon.

    Techniques: In Vitro, Kinase Assay, Recombinant, Autoradiography, Staining, Transfection, Plasmid Preparation, Expressing, Concentration Assay

    PAK1 activation is correlated with tumor stage at presentation. A -Dot plot demonstrating the expression of E-cadherin in the examined tumors in relation to the surgical stage of each tumor. The correlation between variables were examined by Spearman Rank Correlation analysis. B - Dot plot demonstrating the expression of p-PAK1(Thr423) in stage I and stage II/III tumors. The mean between groups was compared by student T-test. C - Dot plot demonstrating the expression of p-Crk-II(Ser41) in stage I and stage II/III tumors. The mean between groups was compared by student T-test.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: PAK1 activation is correlated with tumor stage at presentation. A -Dot plot demonstrating the expression of E-cadherin in the examined tumors in relation to the surgical stage of each tumor. The correlation between variables were examined by Spearman Rank Correlation analysis. B - Dot plot demonstrating the expression of p-PAK1(Thr423) in stage I and stage II/III tumors. The mean between groups was compared by student T-test. C - Dot plot demonstrating the expression of p-Crk-II(Ser41) in stage I and stage II/III tumors. The mean between groups was compared by student T-test.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Activation Assay, Expressing

    PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A -E-cadherin and p-PAK1(Thr423); B -E-cadherin and p-Crk-II(Ser41); C -p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: PAK1 activation and Crk phosphorylation are positively correlated while establish a negative correlation with E-cadherin in NSCLC. Dot plots demonstrating the correlation between A -E-cadherin and p-PAK1(Thr423); B -E-cadherin and p-Crk-II(Ser41); C -p-PAK1(Thr423) and p-Crk-II(Ser41) expression by immunohistochemistry in 17 NSCLC clinical specimens. The average intensity of protein expression across the slide is quantified in a scale from 0-3+. The correlation between the expressions of each two marker was examined by utilizing Spearman Rank Correlation statistical test.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Activation Assay, Expressing, Immunohistochemistry, Marker

    Cooperative disruption of signaling network downstream of mutated KRAS. Schematic view of KRAS/PAK1/Crk signaling pathway is demonstrated. Partial inhibition of KRAS signaling output by tolerable dose of prenylation inhibitors (i.e., FTI + GGTI) in addition to inhibition of a prominent KRAS effector (i.e., PAK1) result in dramatic anti-tumor effects. FTI: farnesyltransferase inhibitor, GGTI: geranylgeranyltransferase inhibitor, PAK1I: p21 associated kinase 1 inhibitor.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: Cooperative disruption of signaling network downstream of mutated KRAS. Schematic view of KRAS/PAK1/Crk signaling pathway is demonstrated. Partial inhibition of KRAS signaling output by tolerable dose of prenylation inhibitors (i.e., FTI + GGTI) in addition to inhibition of a prominent KRAS effector (i.e., PAK1) result in dramatic anti-tumor effects. FTI: farnesyltransferase inhibitor, GGTI: geranylgeranyltransferase inhibitor, PAK1I: p21 associated kinase 1 inhibitor.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Inhibition

    Addition of PAK1 inhibitor to KRAS prenylation inhibitors alters the proliferation of NSCLC cells. Line charts demonstrating mean cell count of H157 and A549 cells upon exposure to prenylation inhibitors [FTI (BMS-225975) and GGTI (P61A6) at 500 nM each], PAK1 inhibitor (FRAX1036 at 10 μM) or combination in comparison to cells exposed to inhibitors’ diluent (DMSO).

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: Addition of PAK1 inhibitor to KRAS prenylation inhibitors alters the proliferation of NSCLC cells. Line charts demonstrating mean cell count of H157 and A549 cells upon exposure to prenylation inhibitors [FTI (BMS-225975) and GGTI (P61A6) at 500 nM each], PAK1 inhibitor (FRAX1036 at 10 μM) or combination in comparison to cells exposed to inhibitors’ diluent (DMSO).

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Cell Counting

    Addition of PAK1 inhibitor to KRAS prenylation inhibitors dramatically alters cell morphology and motility. Photomicrographs of standard wound healing assays in H157 and A549 cells ( KRAS mutant). Cells were exposed to (i) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and geranylgeranyltransferase inhibitor (P61A6 at 2 μM) [F + G]; (ii) PAK1 inhibitor (IPA-3 at 5 μM); (iii) combination of the three inhibitors or (iv) the inhibitor vehicles for 24-48 hours.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: Addition of PAK1 inhibitor to KRAS prenylation inhibitors dramatically alters cell morphology and motility. Photomicrographs of standard wound healing assays in H157 and A549 cells ( KRAS mutant). Cells were exposed to (i) farnesyltransferase inhibitor (BMS-225975 at 2 μM) and geranylgeranyltransferase inhibitor (P61A6 at 2 μM) [F + G]; (ii) PAK1 inhibitor (IPA-3 at 5 μM); (iii) combination of the three inhibitors or (iv) the inhibitor vehicles for 24-48 hours.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Mutagenesis, Indirect Immunoperoxidase Assay

    Addition of PAK1 inhibitor to KRAS prenylation inhibitors dephosphorylated ERK. Western blots demonstrate ERK dephosphorylation following 24 hour exposure of Rh2 cells to (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM), (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM), combination of the two (F + G), or (F + G) along with PAK1 inhibitor (FRAX597 at 40 μM).

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: Addition of PAK1 inhibitor to KRAS prenylation inhibitors dephosphorylated ERK. Western blots demonstrate ERK dephosphorylation following 24 hour exposure of Rh2 cells to (F) farnesyltransferase inhibitor (BMS-225975 at 2 μM), (G) geranylgeranyltransferase inhibitor (P61A6 at 2 μM), combination of the two (F + G), or (F + G) along with PAK1 inhibitor (FRAX597 at 40 μM).

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Western Blot, De-Phosphorylation Assay

    PAK1 activation is inversely correlated with E-cadherin and p120-catenin expression in NSCLC cells. A -Western blots demonstrating E-cadherin, p120-catenin, PAK1 and p-PAK1(Thr423) expression in a panel of NSCLC cells as well as immortalized normal human respiratory epithelial cells (BEAS-2B). B -Bar chart demonstrating signal intensity of p-PAK1/PAK1, p120-catenin and E-cadherin normalized to the value of BEAS-2B cells.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: PAK1 activation is inversely correlated with E-cadherin and p120-catenin expression in NSCLC cells. A -Western blots demonstrating E-cadherin, p120-catenin, PAK1 and p-PAK1(Thr423) expression in a panel of NSCLC cells as well as immortalized normal human respiratory epithelial cells (BEAS-2B). B -Bar chart demonstrating signal intensity of p-PAK1/PAK1, p120-catenin and E-cadherin normalized to the value of BEAS-2B cells.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Activation Assay, Expressing, Western Blot

    KRAS mutant NSCLC specimens express higher activated PAK1 compared to KRAS wild type samples. A -Dot plot demonstrating immunohistochemical expression of p-PAK1(Thr423) in KRAS mutant and KRAS wild type NSCLC clinical specimens. p-PAK1(Thr423) is detectable in all KRAS mutant specimens while most KRAS wild type samples express lower p-PAK1(Thr423). B - Dot plot demonstrating immunohistochemical expression of E-cadherin in KRAS mutant vs. KRAS wild type NSCLC clinical specimens.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: KRAS mutant NSCLC specimens express higher activated PAK1 compared to KRAS wild type samples. A -Dot plot demonstrating immunohistochemical expression of p-PAK1(Thr423) in KRAS mutant and KRAS wild type NSCLC clinical specimens. p-PAK1(Thr423) is detectable in all KRAS mutant specimens while most KRAS wild type samples express lower p-PAK1(Thr423). B - Dot plot demonstrating immunohistochemical expression of E-cadherin in KRAS mutant vs. KRAS wild type NSCLC clinical specimens.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Mutagenesis, Immunohistochemistry, Expressing

    PAK1 activation and Crk phosphorylation are correlated with loss of E-cadherin and p120-catenin in NSCLC specimens. Photomicrographs demonstrating immunohistochemical staining of NSCLC clinical specimens. Samples 12, 54, 16 and 75 harbor high E-cadherin/p120-catenin while expressing no detectable level of p-PAK1(Thr423) and p-Crk-II(Ser41). On the other hand, samples 49, 73 and 57 with detectable p-PAK1(Thr423) and p-Crk-II(Ser41) show very low levels of E-cadherin/p120-catenin.

    Journal: BMC Cancer

    Article Title: Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis

    doi: 10.1186/s12885-015-1360-4

    Figure Lengend Snippet: PAK1 activation and Crk phosphorylation are correlated with loss of E-cadherin and p120-catenin in NSCLC specimens. Photomicrographs demonstrating immunohistochemical staining of NSCLC clinical specimens. Samples 12, 54, 16 and 75 harbor high E-cadherin/p120-catenin while expressing no detectable level of p-PAK1(Thr423) and p-Crk-II(Ser41). On the other hand, samples 49, 73 and 57 with detectable p-PAK1(Thr423) and p-Crk-II(Ser41) show very low levels of E-cadherin/p120-catenin.

    Article Snippet: Interestingly, neither FTI/GGTI nor their combination could alter ERK phosphorylation however addition of PAK1 inhibitor (i.e., FRAX597) to (FTI + GGTI) significantly dephosphorylated ERK (Figure ).

    Techniques: Activation Assay, Immunohistochemistry, Staining, Expressing

    Both PIX and NCK localize to focal adhesions in breast cancer cell lines with active PAK1/2. SK-BR-3 and ZR-75-1 cells were fixed and stained with anti-PIX/anti-rabbit Alexa Fluor 568 or anti-NCK/anti-rabbit Alexa Fluor 488 as described in MATERIALS AND METHODS. Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: Both PIX and NCK localize to focal adhesions in breast cancer cell lines with active PAK1/2. SK-BR-3 and ZR-75-1 cells were fixed and stained with anti-PIX/anti-rabbit Alexa Fluor 568 or anti-NCK/anti-rabbit Alexa Fluor 488 as described in MATERIALS AND METHODS. Bar, 10 μm.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Staining

    Microinjection of a PIX-competing peptide displaces ectopically expressed PAK1 from focal adhesions. SK-BR-3 cells were transfected with EGFP-tagged PAK1 K299R and microinjected with either a PIX competing peptide (PAK1, aa 147–231) or a NCK competing peptide (PAK1, aa 1–74). Fluorescent images were taken every 15 s for 1 h after peptide microinjection or until EGFP PAK was displaced from focal adhesions. Microinjected cells are indicated by an asterisk.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: Microinjection of a PIX-competing peptide displaces ectopically expressed PAK1 from focal adhesions. SK-BR-3 cells were transfected with EGFP-tagged PAK1 K299R and microinjected with either a PIX competing peptide (PAK1, aa 147–231) or a NCK competing peptide (PAK1, aa 1–74). Fluorescent images were taken every 15 s for 1 h after peptide microinjection or until EGFP PAK was displaced from focal adhesions. Microinjected cells are indicated by an asterisk.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Transfection

    Endogenous, activated PAK1/2 localizes to focal adhesions in SK-BR-3 and ZR-75-1 breast cancer cells. MDA-MB-231, SK-BR-3, and ZR-75-1 cells were fixed and stained with anti-phospho-PAK (raised against phosphorylated S199/S204 in PAK1) (anti-pPAK)/anti-rabbit Alexa Fluor 488 and anti-paxillin/anti-mouse Alexa Fluor 568 to visualize phosphorylated, active PAK1/2 and focal adhesions, respectively, as described in MATERIALS AND METHODS.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: Endogenous, activated PAK1/2 localizes to focal adhesions in SK-BR-3 and ZR-75-1 breast cancer cells. MDA-MB-231, SK-BR-3, and ZR-75-1 cells were fixed and stained with anti-phospho-PAK (raised against phosphorylated S199/S204 in PAK1) (anti-pPAK)/anti-rabbit Alexa Fluor 488 and anti-paxillin/anti-mouse Alexa Fluor 568 to visualize phosphorylated, active PAK1/2 and focal adhesions, respectively, as described in MATERIALS AND METHODS.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Multiple Displacement Amplification, Staining

    SK-BR-3 and ZR-75-1 cell lines with activated PAK do not contain detectable levels of active RhoGTPases. Cell lysates with GDP or guanosine 5′- O -(3-thio) triphosphate loading or without nucleotide loading were incubated with GST fusion protein containing the p21 binding domain of PAK1 (GST-PBD). Activated Rac1 (A) or Cdc42 (B) was detected by Western blot analysis of precipitated proteins with monoclonal anti-Rac1 antibody or polyclonal anti-Cdc42 antiserum, respectively. Similar amounts of Rac1 or Cdc42 were detected in cell lysates by Western blot analysis with anti-Rac1 or anti-Cdc42 antibodies. (C) Cell lysates were incubated in the presence or absence of AlF 4 - and incubated with GST fusion protein containing the Rho binding domain of Rhotekin (GST-RBD). Activated RhoA was detected by Western blot analysis of precipitated proteins with monoclonal RhoA antibody. Similar amounts of RhoA in SK-BR-3 and ZR-75-1 cell lysates were detected by Western blotting with anti-RhoA antibody. (D) SK-BR-3 cells were stimulated with 50 ng/ml Heregulin β1 for the indicated times and cell lysates were incubated with GST-PBD and precipitated proteins were detected with anti-Rac1 monoclonal antibody. Equivalent amounts of Rac1 in cell lysates were detected by Western blotting with anti-Rac1.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: SK-BR-3 and ZR-75-1 cell lines with activated PAK do not contain detectable levels of active RhoGTPases. Cell lysates with GDP or guanosine 5′- O -(3-thio) triphosphate loading or without nucleotide loading were incubated with GST fusion protein containing the p21 binding domain of PAK1 (GST-PBD). Activated Rac1 (A) or Cdc42 (B) was detected by Western blot analysis of precipitated proteins with monoclonal anti-Rac1 antibody or polyclonal anti-Cdc42 antiserum, respectively. Similar amounts of Rac1 or Cdc42 were detected in cell lysates by Western blot analysis with anti-Rac1 or anti-Cdc42 antibodies. (C) Cell lysates were incubated in the presence or absence of AlF 4 - and incubated with GST fusion protein containing the Rho binding domain of Rhotekin (GST-RBD). Activated RhoA was detected by Western blot analysis of precipitated proteins with monoclonal RhoA antibody. Similar amounts of RhoA in SK-BR-3 and ZR-75-1 cell lysates were detected by Western blotting with anti-RhoA antibody. (D) SK-BR-3 cells were stimulated with 50 ng/ml Heregulin β1 for the indicated times and cell lysates were incubated with GST-PBD and precipitated proteins were detected with anti-Rac1 monoclonal antibody. Equivalent amounts of Rac1 in cell lysates were detected by Western blotting with anti-Rac1.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Incubation, Binding Assay, Western Blot

    SK-BR-3 and ZR-75-1, but not MDA-MB-231, human breast cancer cells contain constitutively activated PAK1 and/or PAK2. (A) Breast cancer cell lysates were subjected to an in-gel kinase assay by using p47 phox peptide as substrate(see MATERIALS AND METHODS). Western blot analysis of lysates with anti-PAK antiserum demonstrates equal levels of PAK2 in all breast cancer cell lines, with differing levels of PAK1. (B) Cell lysates were immunoprecipitated with anti-PAK antiserum and subjected to an in-gel kinase assay using p47 phox peptide as substrate.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: SK-BR-3 and ZR-75-1, but not MDA-MB-231, human breast cancer cells contain constitutively activated PAK1 and/or PAK2. (A) Breast cancer cell lysates were subjected to an in-gel kinase assay by using p47 phox peptide as substrate(see MATERIALS AND METHODS). Western blot analysis of lysates with anti-PAK antiserum demonstrates equal levels of PAK2 in all breast cancer cell lines, with differing levels of PAK1. (B) Cell lysates were immunoprecipitated with anti-PAK antiserum and subjected to an in-gel kinase assay using p47 phox peptide as substrate.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Multiple Displacement Amplification, Kinase Assay, Western Blot, Immunoprecipitation

    PAK association with PIX and localization to focal adhesions is required for PAK kinase activity. (A) SK-BR-3 cells were microinjected with either a PIX-competing peptide (PAK1, aa 147–231) or a NCK-competing peptide (PAK1, aa 1–74). Thirty minutes after microinjection, cells were fixed and stained with anti-phospho-PAK (anti-pPAK; raised against S199/204 of PAK1)/anti-rabbit Alexa Fluor 488. Cells were scored as described in MATERIALS AND METHODS. Microinjected cells are indicated by an asterisk. (B) SK-BR-3 cells were infected with recombinant Semliki Forest virus constructs encoding myc-tagged PAK1, kinase-dead (K299R), kinase active (T423E), or mutant PAK1 incapable of binding to PIX (R193, P194A). Cell lysates were immunoprecipitated with anti-myc antibodies and subjected to an in-gel kinase assay by using p47 phox peptide as substrate. Immunoprecipitated proteins were subjected to an anti-PAK Western blot to ensure equal loading.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: PAK association with PIX and localization to focal adhesions is required for PAK kinase activity. (A) SK-BR-3 cells were microinjected with either a PIX-competing peptide (PAK1, aa 147–231) or a NCK-competing peptide (PAK1, aa 1–74). Thirty minutes after microinjection, cells were fixed and stained with anti-phospho-PAK (anti-pPAK; raised against S199/204 of PAK1)/anti-rabbit Alexa Fluor 488. Cells were scored as described in MATERIALS AND METHODS. Microinjected cells are indicated by an asterisk. (B) SK-BR-3 cells were infected with recombinant Semliki Forest virus constructs encoding myc-tagged PAK1, kinase-dead (K299R), kinase active (T423E), or mutant PAK1 incapable of binding to PIX (R193, P194A). Cell lysates were immunoprecipitated with anti-myc antibodies and subjected to an in-gel kinase assay by using p47 phox peptide as substrate. Immunoprecipitated proteins were subjected to an anti-PAK Western blot to ensure equal loading.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Activity Assay, Staining, Infection, Recombinant, Construct, Mutagenesis, Binding Assay, Immunoprecipitation, Kinase Assay, Western Blot

    Disruption of the actin cytoskeleton and focal contacts abrogates PAK kinase activity in breast cancer cells with active endogenous PAK1/2. (A) SK-BR-3 and ZR-75–1 cells were treated with dimethyl sulfoxide (0) or 2 μM cytochalasin D (cyto D) for 10 and 60 min. Cells were lysed and subjected to an in-gel kinase assay by using p47 phox peptide as substrate. (B) SK-BR-3 and ZR-75-1 cells were untreated (att), treated with 2 μM cytochalasin D (cyto D) for 60 min, or trypsinized and placed in suspension for 30 min (susp) in PBS before lysis and in gel kinase assay. Levels of PAK did not change with treatment, as detected by immunoblot with anti-PAK antisera.

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions D⃞

    doi: 10.1091/mbc.E03-08-0604

    Figure Lengend Snippet: Disruption of the actin cytoskeleton and focal contacts abrogates PAK kinase activity in breast cancer cells with active endogenous PAK1/2. (A) SK-BR-3 and ZR-75–1 cells were treated with dimethyl sulfoxide (0) or 2 μM cytochalasin D (cyto D) for 10 and 60 min. Cells were lysed and subjected to an in-gel kinase assay by using p47 phox peptide as substrate. (B) SK-BR-3 and ZR-75-1 cells were untreated (att), treated with 2 μM cytochalasin D (cyto D) for 60 min, or trypsinized and placed in suspension for 30 min (susp) in PBS before lysis and in gel kinase assay. Levels of PAK did not change with treatment, as detected by immunoblot with anti-PAK antisera.

    Article Snippet: Cells grown on coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, permeabilized in PBS containing 0.5% Triton X-100 for 20 min, and blocked with 3% BSA in PBS for 1 h. Coverslips were incubated for 1 h with one of the following primary antibodies: polyclonal αpPAK raised against the phosphorylated peptide SKRST(P)MVGTPYC corresponding to amino acids 419–429 of PAK1 at 1:200 , polyclonal αpPAK raised against the phosphorylated peptide PEHTKS(P)VYTRS(P)VIEP corresponding to amino acids 193–207 of PAK1 at 1:500 , monoclonal αvinculin (Sigma-Aldrich, St. Louis, MO) at 1:1000 and monoclonal αpaxillin at 1:500 (BD Transduction Laboratories, Lexington, KY), polyclonal αPIX at 1:2000, polyclonal azyxin B71 at 1:500 , and polyclonal αNCK (NeoMarkers) at 1:200.

    Techniques: Activity Assay, Kinase Assay, Lysis